Trans-cardial perfusion, sagittal slice preparation, modified aCSF solution as well as addition of neuro-protectants have greatly increased slice viability. Co-application of KA and CCh induced persistent theta and gamma oscillations. This dual rhythm was identified to be led by S1 in an intact slice but both M1 and S1 have individual rhythm generators. These nested oscillations seem to have distinct mechanisms and were identified to be coupled from PAC analysis. However, the percentage of coupling varied in various types of slice preparations with high modulation index (MI) in M1 only slice followed by S1 only slice when compared to intact slice preparations.
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4 Characterisation of pharmacologically
induced delta oscillations in LV of primary
motor cortex (M1)
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4.1 Introduction
4.1.1 Neurochemical aspects of the sleep-wake cycle
The sleep-wake cycle is associated with electrical and chemical changes that enable the transition between various sleep and awake states. However, overall orchestrated co- ordination of these electrical and chemical processes in specific brain regions have yet to be explored. We now know that neurons in various regions in the brain such as hypothalamus and brain stem undergo transitions between states via regulated release of various neuromodulators in cortex and thalamus and these are discussed below:
Acetylcholine (ACh) has been identified to play a role in both sleep and awake states. Cholinergic neurons fire at high frequency in the awake state whilst showing no activity in REM sleep. The NREM state has been shown to have an intermediate cholinergic tone (Hobson et al., 2003; Espana and Scammell, 2004; Jones, 2005). Interestingly, ACh neurons in pedunculopontine (PPN), have been identified to have high firing rates both in wake and REM sleep (Wake-ON) more in REM than wake or non-REM sleep (REM-ON) in which the increased firing was observed 20-60 s prior to state change. This shift in patterns has been suggested to underpin the role of PPN in desynchronizing the cortex during awake and REM states (Steriade et al., 1990).
Dopamine (DA) plays a key role in arousal and sleep states. DA has been implicated in awake-associated behaviours such as movement, cognition and arousal as lesioning dopaminergic innervations has shown to decrease arousal. In Parkinson’s, a neuro- degenerative disease with symptoms caused by depletion of DA, sufferers show difficulty in active awake-state maintenance. CNS stimulants such as amphetamines and cocaine act on dopaminergic systems which enhances arousal, increased vigilance and attention either by stimulating DA release or by inhibiting re-uptake. Interestingly, compounds like caffeine, that does not act via DA systems, may also influence DA release in the synaptic cleft (Schultz and Miller, 2004; Boutrel and Koob, 2004; Espana and Scammell, 2004; Wisor, 2005; Jones, 2005). Despite the above evidence that DA is critical for arousal, it has not traditionally been grouped with the reticular activating system based on the fact that DA neurons fire at equal rates both in awake and sleep states (Trulson et al., 1981).
Histamine (HA) was found to be wake promoting from the fact that histaminergic neurons fire at higher rates in awake state while remaining silent during REM and NREM sleep (Takahashi et al., 2006), which was revealed in electrophysiological recordings. Furthermore, increased sleepiness is an unwanted side effect of some anti-histamines (Hobson et al., 2003; Jones, 2005).
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Serotonin (5-HT) also has been implicated in wake promoting since serotonergic cells fire at a maximum rate in awake state with progressive decline in activity in NREM sleep and no activity in REM sleep. However, the activity was found to be resumed prior to the end of REM episodes (McGinty and Harper, 1976; Trulson and Jacobs, 1979). Although evidence supports the role of 5-HT in the awake state, there is controversy over its role in sleep (Jones, 2005; Espana and Scammell, 2004). Studies suggest this confusion is probably due to many receptor subtypes as well as many activities attributed to 5-HT (Espana and Scammell, 2004; Jones, 2005).
GABA concentration was found to be the highest in cortex during NREM sleep and is reduced in REM sleep in comparison to awake state (NREM>awake>REM) (Vanini et al., 2011). Increased levels of extracellular GABA may indeed enhance synaptic inhibition whereby balancing synaptic inhibition and excitation in slow wave sleep (Haider et al., 2006; Rudolph et al., 2007). However, several studies have identified co-release of GABA with ACh and HA suggesting decreased levels of GABA in NREM sleep (Saunders et al., 2015; Yu et al., 2015).
4.1.2 Significance of delta oscillations
Delta oscillations have been implicated both in deep sleep state and awake states performing cognitive tasks. This apparent contradiction was explained by the existence of some type of inhibition during task that would inhibit the non-relevant neuronal activity (Vogel et al., 1968) known as behavioural inhibition (Kamarajan et al., 2004; Knyazev, 2007; Putman, 2011). Behavioural studies on humans showed that delta oscillations which are prevalent in deep sleep may mediate an enhancement in declarative memory (Huber et al., 2004; Aeschbach, 2009). Rotation adaption tasks on human subjects performed in this study showed a greater correlation between increases in the 1-4 frequency range and improved task performance after sleeping in EEG of cerebral cortex (Huber et al., 2004). Sleep has been suggested to facilitate memory consolidation by strengthening and stabilising memories (Gais et al., 2000; Stickgold et al., 2000; Maquet, 2001; Walker and Stickgold, 2006; Rasch and Born, 2013). It was demonstrated that better performance was observed in a group that were allowed to sleep after learning compared to a group that were awake after learning. Besides consolidation of previously acquired memories sleep also plays a role in acquisition of new memories (Van Der Werf et al., 2009) which was proved in a study where short period of sleep preceding learning has enabled a greater capacity to encode information (Mander et al., 2011). Also, it has been reported that the synaptic plasticity associated with low frequency oscillations during sleep contribute to memory consolidation processes (Steriade and Timofeev, 2003). Interestingly, some studies have suggested a role for the phase component of delta oscillations in decision making and signal detection (Basar-Eroglu et al., 1992) by rhythmically modulating the rate of sensory
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information received over time (Wyart et al., 2012) and in many cognitive processes (Basar et al., 2001). Delta oscillations are also known to play a critical role in pathological conditions: decreased synchronous delta activity was observed in schizophrenic patients (Ford et al., 2008), reduced delta amplitude and coherence in Alzheimer’s patients (Guntekin et al., 2008; Yener and Basar et al., 2010) increased delta power in Parkinson`s patients under medication (Kryzhanovskii et al., 1990) and disrupted delta activity in epilepsy (Tao et al., 2011; for review see Harmony, 2013).
Despite its functional significance, cellular mechanisms of cortical delta rhythmogenesis and its mechanistic origin have not yet been explored. There is a hypothesis that the cortical delta rhythm was mediated by intrinsically bursting neurons (Amzica and Steriade, 1998) however, in that study the usage of depolarising current pulse (intercellular injections) did not match with long lasting hyperpolariaation conditions during sleep reported in cortex (Steriade et al., 2001; Timofeev et al., 2000). This leads to further discrepancies regarding the mechanism underlying cortical delta rhythm. A recent study in slices containing secondary somatosensory/parietal area (S2/Par2) has revealed generation of delta oscillations in vitro by maintaining neuro-modulatory state equivalent to deep sleep state (Carracedo et al., 2013). Our current study performed on LV of M1 demonstrates the generation of delta oscillations by pharmacological manipulations and investigates the contribution of various receptors involved in the rhythm generation and the pharmacological equivalent awake state. Characterisation of delta rhythm in control slice preparations may facilitate understanding of the mechanisms underlying various pathological conditions like Alzheimer’s disease.
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