Francisco J. Urbano

Temporal binding via cortical coincidence detection of specific and nonspecific thalamocortical inputs: A voltage-dependent dye-imaging study in mouse brain slices

Voltage-sensitive dye imaging of mouse thalamocortical slices demonstrated that electrical stimulation of the centrolateral intralaminar thalamic nucleus (CL) resulted in the specific activation of thalamic reticular nucleus, striatum/putamen, and cortical layers 5, 6, and 1. By contrast, ventrobasal (VB) thalamic stimulation, while activating the reticular and basal ganglia nuclei, also activated directly layers 4 and deep 5 of the cortex. Conjoined stimulation of the VB and CL nuclei resulted in supralinear summation of the two inputs at cortical output layer 5, demonstrating coincidence detection along the apical dendrites. This supralinear summation was also noticed at gamma band stimulus frequency (≈40 Hz). Direct stimulation of cortical layer 1, after a radial section of the cortex that spared only that layer, was shown to sum supralinearly with the cortical activation triggered by VB stimulation, providing a second demonstration for coincidence detection. Coincidence detection by coactivation of the specific (VB) and nonspecific (CL) thalamic nuclei has been proposed as the basis for the temporal conjunction that supports cognitive binding in the brain.

Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca2+ channels

Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N- and R-type Ca2+ channels when the P/Q-type channel α1A subunit was deleted. R-type channels lay close to Ca2+ sensors for exocytosis and IK(Ca) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca2+ channels in the hierarchy P/Q>R>N. The α1A−/− neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca2+/Mg2+ ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.

Modafinil enhances thalamocortical activity by increasing neuronal electrotonic coupling.

Modafinil (Provigil, Modiodal), an antinarcoleptic and mood-enhancing drug, is shown here to sharpen thalamocortical activity and to increase electrical coupling between cortical interneurons and between nerve cells in the inferior olivary nucleus. After irreversible pharmacological block of connexin permeability (i.e., by using either 18β-glycyrrhetinic derivatives or mefloquine), modafinil restored electrotonic coupling within 30 min. It was further established that this restoration is implemented through a Ca2+/calmodulin protein kinase II-dependent step.

γ-Band deficiency and abnormal thalamocortical activity in P/Q-type channel mutant mice

Thalamocortical in vivo and in vitro function was studied in mice lacking P/Q-type calcium channels (CaV2.1), in which N-type calcium channels (CaV2.2) supported central synaptic transmission. Unexpectedly, in vitro patch recordings from thalamic neurons demonstrated no γ-band subthreshold oscillation, and voltage-sensitive dye imaging demonstrated an absence of cortical γ-band-dependent columnar activation involving cortical inhibitory interneuron activity. In vivo electroencephalogram recordings showed persistent absence status and a dramatic reduction of γ-band activity. Pharmacological block of T-type calcium channels (CaV3), although not noticeably affecting normal control animals, left the knockout mice in a coma-like state. Hence, although N-type calcium channels can rescue P/Q-dependent synaptic transmission, P/Q calcium channels are essential in the generation of γ-band activity and resultant cognitive function.

Mechanism behind gamma band activity in the pedunculopontine nucleus

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low‐amplitude, high‐frequency oscillatory activity in the beta–gamma band range (∼20–80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage‐gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8–14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage‐clamped at holding potentials above −30 mV, suggesting that their origin may be spatially located beyond voltage‐clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N‐type calcium channel blocker ω‐conotoxin‐GVIA partially reduced gamma oscillations, while the P/Q‐type blocker ω‐agatoxin‐IVA abolished them. Both ω‐CgTX and ω‐Aga blocked voltage‐dependent calcium currents, by 56 and 52% respectively. The delayed rectifier‐like potassium channel blocker α‐dendrotoxin also abolished gamma oscillations. In carbachol‐induced PPN population responses, ω‐agatoxin‐IVA reduced higher, and ω‐CgTx mostly lower, frequencies. These results suggest that voltage‐dependent P/Q‐ and, to a lesser extent, N‐type calcium channels mediate gamma oscillations in PPN.

Calcium channels involved in neurotransmitter release at adult, neonatal and P/Q-type deficient neuromuscular junctions (Review).

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I Ca ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca 2+ inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca 2+ -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.

Coherence and frequency in the reticular activating system (RAS)

This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit (1) electrical coupling mainly in GABAergic cells, and (2) gamma band activity in virtually all of the cells. Specifically, cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine dorsal subcoeruleus nucleus dorsalis (SubCD) (1) show electrical coupling, and (2) all fire in the beta/gamma band range when maximally activated, but no higher. The mechanism behind electrical coupling is important because the stimulant modafinil was shown to increase electrical coupling. We also provide recent findings demonstrating that all cells in the PPN and Pf have high threshold, voltage-dependent P/Q-type calcium channels that are essential to gamma band activity. On the other hand, all SubCD, and some PPN, cells manifested sodium-dependent subthreshold oscillations. A novel mechanism for sleep-wake control based on transmitter interactions, electrical coupling, and gamma band activity is described. We speculate that continuous sensory input will modulate coupling and induce gamma band activity in the RAS that could participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions.

Gamma Band Activity in the Reticular Activating System

This review considers recent evidence showing that cells in three regions of the reticular activating system (RAS) exhibit gamma band activity, and describes the mechanisms behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine subcoeruleus nucleus dorsalis (SubCD) all fire in the beta/gamma band range when maximally activated, but no higher. The mechanisms behind this ceiling effect have been recently elucidated. We describe recent findings showing that every cell in the PPN have high-threshold, voltage-dependent P/Q-type calcium channels that are essential, while N-type calcium channels are permissive, to gamma band activity. Every cell in the Pf also showed that P/Q-type and N-type calcium channels are responsible for this activity. On the other hand, every SubCD cell exhibited sodium-dependent subthreshold oscillations. A novel mechanism for sleep–wake control based on well-known transmitter interactions, electrical coupling, and gamma band activity is described. The data presented here on inherent gamma band activity demonstrates the global nature of sleep–wake oscillation that is orchestrated by brainstem–thalamic mechanism, and questions the undue importance given to the hypothalamus for regulation of sleep–wakefulness. The discovery of gamma band activity in the RAS follows recent reports of such activity in other subcortical regions like the hippocampus and cerebellum. We hypothesize that, rather than participating in the temporal binding of sensory events as seen in the cortex, gamma band activity manifested in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking and paradoxical sleep. Most of our thoughts and actions are driven by pre-conscious processes. We speculate that continuous sensory input will induce gamma band activity in the RAS that could participate in the processes of pre-conscious awareness, and provide the essential stream of information for the formulation of many of our actions.

Acute modulation of calcium currents and synaptic transmission by gabapentinoids.

Gabapentin and pregabalin are anticonvulsant drugs that are extensively used for the treatment of several neurological and psychiatric disorders. Gabapentinoids (GBPs) are known to have a high affinity binding to α2δ-1 and α2δ-2 auxiliary subunit of specific voltage-gated calcium channels. Despite the confusing effects reported on Ca2+ currents, most of the studies showed that GBPs reduced release of various neurotransmitters from synapses in several neuronal tissues. We showed that acute in vitro application of pregabalin can reduce in a dose dependent manner synaptic transmission in both neuromuscular junctions and calyx of Held-MNTB excitatory synapses. Furthermore presynaptic Ca2+ currents treated with pregabalin are reduced in amplitude, do not show inactivation at a clinically relevant low concentration of 100 μM and activate and deactive faster. These results suggest novel modulatory role of acute pregabalin that might contribute to better understanding its anticonvulsant/analgesic clinical effects.

Modafinil Abrogates Methamphetamine-Induced Neuroinflammation and Apoptotic Effects in the Mouse Striatum

Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4×5 mg/kg, i.p., 2 h apart) and modafinil co-administration (2×90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections) on glial cells (microglia and astroglia). We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.