Divergent behavioral responses in protracted opioid withdrawal in male and female C57BL/6J mice.

Persons suffering from opioid use disorder (OUD) experience long-lasting dysphoric symptoms well into extended periods of withdrawal. This protracted withdrawal syndrome is notably characterized by heightened anxiety and hyperkatifeia. Here, we investigated if an exacerbated withdrawal model of acute morphine dependence results in lasting behavioral adaptation 6 weeks into forced abstinence in C57BL/6J mice. We found that our exacerbated morphine withdrawal paradigm produced distinct alterations in behavior in elevated plus maze (EPM), open field, and social interaction tests in male and female mice. Following protracted withdrawal male mice showed enhanced exploration of the open arms of the EPM, reduced latency to enter the corner of the OF, and a social interaction deficit. In contrast, female mice showed enhanced thigmotaxis in the OF. In both sexes, protracted withdrawal enhanced locomotor behavior in response to subsequent morphine challenge, albeit at different doses. These findings will be relevant for future investigation examining the neural mechanisms underlying these behaviors and will aid in uncovering physiological sex differences in response to opioid withdrawal.

Inhibitory transmission in the bed nucleus of the stria terminalis in male and female mice following morphine withdrawal.

The United States is experiencing an opioid crisis imposing enormous fiscal and societal costs and driving the staggering overdose death rate. While prescription opioid analgesics are essential for treating acute pain, cessation of use in individuals with a physical dependence induces an aversive withdrawal syndrome that promotes continued drug use to alleviate/avoid these symptoms. Additionally, repeated bouts of withdrawal often lead to an increased propensity for relapse. Understanding the neurobiology underlying withdrawal is essential for providing novel treatment options to alleviate physiological and affective components accompanying the cessation of opiate use. Here, we administered morphine and precipitated withdrawal with naloxone to investigate behavioral and cellular responses in C57BL/6J male and female mice. Following 3 days of administration, both male and female mice demonstrated sensitized withdrawal symptoms. Since the bed nucleus of the stria terminalis (BNST) plays a role in mediating withdrawal-associated behaviors, we examined plastic changes in inhibitory synaptic transmission within this structure 24 hours following the final precipitated withdrawal. In male mice, morphine withdrawal increased spontaneous GABAergic signaling compared with controls. In contrast, morphine withdrawal decreased spontaneous GABAergic signaling in female mice. Intriguingly, these opposing GABAergic effects were contingent upon activity-dependent dynamics within the ex vivo slice. Our findings suggest that male and female mice exhibit some divergent cellular responses in the BNST following morphine withdrawal, and alterations in BNST inhibitory signaling may contribute to the expression of behaviors following opioid withdrawal.

Modulation of gamma oscillations in the pedunculopontine nucleus by neuronal calcium sensor protein-1: relevance to schizophrenia and bipolar disorder.

Reduced levels of gamma-band activity are present in schizophrenia and bipolar disorder patients. In the same disorders, increased neuronal calcium sensor protein-1 (NCS-1) expression was reported in a series of postmortem studies. These disorders are also characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). The discovery of gamma-band activity in the pedunculopontine nucleus (PPN), the cholinergic arm of the RAS, revealed that such activity was mediated by high-threshold calcium channels that are regulated by NCS-1. We hypothesized that NCS-1 normally regulates gamma-band oscillations through these calcium channels and that excessive levels of NCS-1, such as would be expected with overexpression, decrease gamma-band activity. We found that PPN neurons in rat brain slices manifested gamma-band oscillations that were increased by low levels of NCS-1 but suppressed by high levels of NCS-1. Our results suggest that NCS-1 overexpression may be responsible for the decrease in gamma-band activity present in at least some schizophrenia and bipolar disorder patients.

Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types.

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV. Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN).

The pedunculopontine nucleus (PPN) is part of the Reticular Activating System, and active during waking and REM sleep. Previous results showed that all PPN cells plateau at gamma frequencies and intrinsic membrane oscillations in PPN neurons are mediated by high-threshold N- and P/Q-type Ca(2+) channels. The present study was designed to determine whether some PPN cells have only N-, only P/Q-, or both N- and P/Q-type Ca(2+) channels. We used patch-clamp recordings in PPN cells in slices from anesthetized rat pups in the presence of synaptic receptor blockers (SB) and Tetrodotoxin (TTX), and applied ramps to induce intrinsic membrane oscillations. We found that all PPN cell types showed gamma oscillations in the presence of SB+TTX when using current ramps. In 50% of cells, the N-type Ca(2+) channel blocker ω-Conotoxin-GVIA (ω-CgTx) reduced gamma oscillation amplitude, while subsequent addition of the P/Q-type blocker ω-Agatoxin-IVA (ω-Aga) blocked the remaining oscillations. Another 20% manifested gamma oscillations that were not significantly affected by the addition of ω-CgTx, however, ω-Aga blocked the remaining oscillations. In 30% of cells, ω-Aga had no effect on gamma oscillations, while ω-CgTx blocked them. These novel results confirm the segregation of populations of PPN cells as a function of the calcium channels expressed, that is, the presence of cells in the PPN that manifest gamma band oscillations through only N-type, only P/Q-type, and both N-type and P/Q-type Ca(2+) channels.

Pedunculopontine arousal system physiology - Deep brain stimulation (DBS).

This review describes the wake/sleep symptoms present in Parkinson׳s disease, and the role of the pedunculopontine nucleus in these symptoms. The physiology of PPN cells is important not only because it is a major element of the reticular activating system, but also because it is a novel target for deep brain stimulation in the treatment of gait and postural deficits in Parkinson׳s disease. A greater understanding of the physiology of the target nuclei within the brainstem and basal ganglia, amassed over the past decades, has enabled increasingly better patient outcomes from deep brain stimulation for movement disorders.

Pedunculopontine arousal system physiology - Implications for insomnia.

We consider insomnia a disorder of waking rather than a disorder of sleep. This review examines the role of the reticular activating system, especially the pedunculopontine nucleus, in the symptoms of insomnia, mainly representing an overactive waking drive. We determined that high frequency activity during waking and REM sleep is controlled by two different intracellular pathways and channel types in PPN cells. We found three different PPN cell types that have one or both channels and may be active during waking only, REM sleep only, or both. These discoveries point to a specific mechanism and novel therapeutic avenues for insomnia.

Pedunculopontine arousal system physiology-Effects of psychostimulant abuse.

This review describes the interactions between the pedunculopontine nucleus (PPN), the ventral tegmental area (VTA), and the thalamocortical system. Experiments using modulators of cholinergic receptors in the PPN clarified its role on psychostimulant-induced locomotion. PPN activation was found to be involved in the animal's voluntary search for psychostimulants. Every PPN neuron is known to generate gamma band oscillations. Voltage-gated calcium channels are key elements in the generation and maintenance of gamma band activity of PPN neurons. Calcium channels are also key elements mediating psychostimulant-induced alterations in the thalamic targets of PPN output. Thus, the PPN is a key substrate for maintaining arousal and REM sleep, but also in modulating psychostimulant self-administration.

Pedunculopontine Gamma Band Activity and Development.

This review highlights the most important discovery in the reticular activating system in the last 10 years, the manifestation of gamma band activity in cells of the reticular activating system (RAS), especially in the pedunculopontine nucleus, which is in charge of waking and rapid eye movement (REM) sleep. The identification of different cell groups manifesting P/Q-type Ca(2+) channels that control waking vs. those that manifest N-type channels that control REM sleep provides novel avenues for the differential control of waking vs. REM sleep. Recent discoveries on the development of this system can help explain the developmental decrease in REM sleep and the basic rest-activity cycle.

Pedunculopontine Nucleus Gamma Band Activity-Preconscious Awareness, Waking, and REM Sleep.

The pedunculopontine nucleus (PPN) is a major component of the reticular activating system (RAS) that regulates waking and REM sleep, states of high-frequency EEG activity. Recently, we described the presence of high threshold, voltage-dependent N- and P/Q-type calcium channels in RAS nuclei that subserve gamma band oscillations in the mesopontine PPN, intralaminar parafascicular nucleus (Pf), and pontine subcoeruleus nucleus dorsalis (SubCD). Cortical gamma band activity participates in sensory perception, problem solving, and memory. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. That is, the RAS may play an early permissive role in volition. Our latest results suggest that (1) the manifestation of gamma band activity during waking may employ a separate intracellular pathway compared to that during REM sleep, (2) neuronal calcium sensor (NCS-1) protein, which is over expressed in schizophrenia and bipolar disorder, modulates gamma band oscillations in the PPN in a concentration-dependent manner, (3) leptin, which undergoes resistance in obesity resulting in sleep dysregulation, decreases sodium currents in PPN neurons, accounting for its normal attenuation of waking, and (4) following our discovery of electrical coupling in the RAS, we hypothesize that there are cell clusters within the PPN that may act in concert. These results provide novel information on the mechanisms controlling high-frequency activity related to waking and REM sleep by elements of the RAS.