Optogenetic activation of the inhibitory nigro-collicular circuit evokes contralateral orienting movements in mice.

We report that increasing inhibition from the basal ganglia (BG) to the superior colliculus (SC) through the substantia nigra pars reticulata (nigra) using in vivo optogenetic activation of GABAergic terminals in mice produces contralateral orienting movements. These movements are unexpected because decreases, and not increases, in nigral activity are generally associated with the initiation of orienting movements. We found that, in slice recordings, the same optogenetic stimulation of nigral terminals producing movements in vivo evokes post-inhibitory rebound depolarization followed by Na+ spikes in SC output neurons. Moreover, blocking T-type Ca2+ channels in slices prevent post-inhibitory rebound and subsequent Na+ spiking in SC output neurons and also reduce the likelihood of contralateral orienting in vivo. On the basis of these results, we propose that, in addition to the permissive role, the BG may play an active role in the generation of orienting movements in mice by driving post-inhibitory rebound depolarization in SC output neurons.

Parvalbumin and GABA Microcircuits in the Mouse Superior Colliculus.

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.

Asynchronous ripple oscillations between left and right hippocampi during slow-wave sleep.

Spatial memory, among many other brain processes, shows hemispheric lateralization. Most of the published evidence suggests that the right hippocampus plays a leading role in the manipulation of spatial information. Concurrently in the hippocampus, memory consolidation during sleep periods is one of the key steps in the formation of newly acquired spatial memory traces. One of the most characteristic oscillatory patterns in the hippocampus are sharp-wave ripple (SWR) complexes. Within this complex, fast-field oscillations or ripples have been demonstrated to be instrumental in the memory consolidation process. Since these ripples are relevant for the consolidation of memory traces associated with spatial navigation, and this process appears to be lateralized, we hypothesize that ripple events between both hippocampi would exhibit different temporal dynamics. We tested this idea by using a modified "split-hyperdrive" that allows us to record simultaneous LFPs from both right and left hippocampi of Sprague-Dawley rats during sleep. We detected individual events and found that during sleep periods these ripples exhibited a different occurrence patterns between hemispheres. Most ripple events were synchronous between intra- rather than inter-hemispherical recordings, suggesting that ripples in the hippocampus are independently generated and locally propagated within a specific hemisphere. In this study, we propose the ripples' lack of synchrony between left and right hippocampi as the putative physiological mechanism underlying lateralization of spatial memory.

Essential role for phosphatidylinositol 4,5-bisphosphate in the expression, regulation, and gating of the slow afterhyperpolarization current in the cerebral cortex.

Many neurons of the CNS and peripheral nervous system express a slow afterhyperpolarization that is mediated by a slow calcium-activated potassium current. Previous work has shown that this aftercurrent regulates repetitive firing and is an important target for neuromodulators signaling through receptors coupled to G-proteins of the Gα(q-11) and Gα(s) subtypes. Yet, despite considerable effort, a molecular-level understanding of the potassium current underlying the slow afterhyperpolarization and its modulation has proven elusive. Here, we use a combination of pharmacological and molecular biological approaches in cortical brain slices to show that the functional expression of the slow calcium-activated afterhyperpolarizing current in pyramidal cells is critically dependent on membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and that this dependence accounts for its inhibition by 5-HT(2A) receptors. Furthermore, we show that PtdIns(4,5)P(2) regulates the calcium sensitivity of I(sAHP) in a manner that suggests it acts downstream from the rise in intracellular calcium. These results clarify key functional aspects of the slow afterhyperpolarization current and its modulation by 5-HT(2A) receptors and point to a key role for PtdIns(4,5)P(2) in the gating of this current.

Visinin-like neuronal calcium sensor proteins regulate the slow calcium-activated afterhyperpolarizing current in the rat cerebral cortex.

Many neurons in the nervous systems express afterhyperpolarizations that are mediated by a slow calcium-activated potassium current. This current shapes neuronal firing and is inhibited by neuromodulators, suggesting an important role in the regulation of neuronal function. Surprisingly, very little is currently known about the molecular basis for this current or how it is gated by calcium. Recently, the neuronal calcium sensor protein hippocalcin was identified as a calcium sensor for the slow afterhyperpolarizing current in the hippocampus. However, while hippocalcin is very strongly expressed in the hippocampus, this protein shows a relatively restricted distribution in the brain. Furthermore, the genetic deletion of this protein only partly reduces the slow hyperpolarizing current in hippocampus. These considerations question whether hippocalcin can be the sole calcium sensor for the slow afterhyperpolarizing current. Here we use loss of function and overexpression strategies to show that hippocalcin functions as a calcium sensor for the slow afterhyperpolarizing current in the cerebral cortex, an area where hippocalcin is expressed at much lower levels than in hippocampus. In addition we show that neurocalcin δ, but not VILIP-2, can also act as a calcium sensor for the slow afterhyperpolarizing current. Finally we show that hippocalcin and neurocalcin δ both increase the calcium sensitivity of the afterhyperpolarizing current but do not alter its sensitivity to inhibition by carbachol acting through the Gαq-11-PLCß signaling cascade. These results point to a general role for a subgroup of visinin-like neuronal calcium sensor proteins in the activation of the slow calcium-activated afterhyperpolarizing current.

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 Galpha(q-11) dominant negative and is also markedly reduced in a phospholipase C beta1 (PLCbeta1) 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 Galpha(q-11)/PLCbeta1 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.

Serotonergic regulation of calcium-activated potassium currents in rodent prefrontal cortex.

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.

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 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.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and structurally related phenylethylamines are potent 5-HT2A receptor antagonists in Xenopus laevis oocytes.

1. We recently described that several 2-(2,5-dimethoxy-4-substituted phenyl)ethylamines (PEAs), including 4-I=2C-I, 4-Br=2C-B, and 4-CH(3)=2C-D analogs, are partial agonists at 5-HT(2C) receptors, and show low or even negligible intrinsic efficacy at 5-HT(2A) receptors. These results raised the proposal that these drugs may act as 5-HT(2) antagonists. 2. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5-HT(2A) or 5-HT(2C) receptors. The above-mentioned PEAs and its 4-H analog (2C-H) blocked the 5-HT-induced currents at 5-HT(2A), but not at the 5-HT(2C) receptor, revealing 5-HT(2) receptor subtype selectivity. The 5-HT(2A) receptor antagonism required a 2-min preincubation to attain maximum inhibition. 3. All PEAs tested shifted the 5-HT concentration-response curves to the right and downward. Their potencies varied with the nature of the C(4) substituent; the relative rank order of their 5-HT(2A) receptor antagonist potency was 2C-I>2C-B>2C-D>2C-H. 4. The present results demonstrate that in X. laevis oocytes, a series of 2,5-dimethoxy-4-substituted PEAs blocked the 5-HT(2A) but not the 5-HT(2C) receptor-mediated responses. As an alternative hypothesis, we suggest that the psychostimulant activity of the PEAs may not be exclusively associated with partial or full 5-HT(2A) receptor agonism.

Differences in potency and efficacy of a series of phenylisopropylamine/phenylethylamine pairs at 5-HT(2A) and 5-HT(2C) receptors.

The pharmacological profile 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 efficacy and relative potency of these drugs were determined and compared to classical 5-HT(2) receptor agonists and antagonists. The rank order of agonist potency at the 5-HT(2A) receptor was: alpha-methyl-5-HT=5-HT>m-CPP>MK-212; at the 5-HT(2C) receptor the order was: 5-HT>alpha-methyl-5-HT>MK-212>m-CPP. All these compounds were full agonists at the 5-HT(2C) receptor, but alpha-methyl-5-HT and m-CPP showed lower efficacy at the 5-HT(2A) receptor. 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, confirming their relative receptor selectivities. 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 efficacies. All these drugs had higher efficacies at 5-HT(2C) receptors; DOI was a full 5-HT(2C) agonist. 2C-I and the other phenylethylamines examined showed relative efficacies at the 5-HT(2C) receptor ranging from 44+/-10% to 76+/-16%. 2C-N was a 5-HT(2) receptor antagonist; the mechanism was competitive at the 5-HT(2A), but non-competitive 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. The alpha-methyl group determines the efficacy of these phenylalkylamines at the 5-HT(2A) and 5-HT(2C) receptors.

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors.

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.