Alpha2-AR agonists improve spatial memory

The most striking finding from the object recognition experiments was that guanfacine selectively improved spatial memory in NK1R-/- mice, whereas recognition memory in both genotypes, and spatial memory in wildtypes, remained unaffected. In contrast, medetomidine improved spatial memory in wildtypes only: a response that was only evident at the lowest

Chapter 4

120 dose (1 g/kg). This confirms that both 2-AR agonists improve spatial memory, albeit at specific doses and in a genotype-dependent manner. Even though the test used here is, strictly speaking, one of short-term spatial memory rather than spatial working memory per se, the two may be close enough in nature to show comparable changes. 2-AR agonists do improve spatial working memory (SWM): for example, in tests of vigilance and working memory, activation of α2A-ARs enhances performance of both rats and monkeys in delayed-alternation (Carlson et al. 1992) and delayed-response tasks (Arnsten et al. 1988). However, this is not always replicable: guanfacine failed to improve SWM in one study of aged non-human primates (Decamp et al. 2011). These effects are likely to be mediated by 2-ARs, rather than 1-ARs, because 1-AR agonists, administered systemically or by local infusion into dlPFC, impair SWM in monkeys (Arnsten and Jentsch 1997; Mao et al. 1999).

The differences between guanfacine and medetomidine reported here could be due to their subtype-selectivity: guanfacine is selective for the 2A-AR subtype (Kd values of 19.9 and 344nM for 2A- and 2C-AR subtypes, respectively) (Uhlen and Wikberg 1991; Uhlen et al. 1992), whereas medetomidine is non-subtype selective. It seems the effects of 2-AR agonists on SWM are due to actions at the 2A-AR subtype: improvements in spatial working memory in rhesus monkeys are reversed by the 2A-AR antagonist, idazoxan (Franowicz and Arnsten 2002; Arnsten and Jin 2012; Kim et al. 2012), and mice with functional ablation of α2A-ARs display deficits in working memory, which are not relieved by guanfacine (Franowicz et al. 2002). However, in contrast, 2C-AR -knockout mice also display deficits in spatial memory (Bjorklund et al. 2001), but this is improved by dexmedetomidine (Tanila et al. 1999; Bjorklund

et al. 2001). Similarly, 2C-AR overexpression impairs water maze learning in mice, and this is alleviated by non-subtype selective 2-AR antagonists (Bjorklund et al.

1998; Bjorklund et al. 2000). Together this evidence suggests that the

2C-AR subtype can modulate SWM, but this subtype is not necessary for the SWM-enhancing effects of adrenoceptor agents.

Another explanation for the difference between the two agonists could lie in their affinities for imidazoline receptors (I1 and I2). The

Figure 4.7 – Chemical structure of the imidazole ring

Chapter 4

121 structure of medetomidine contains the imidazole ring (see Figure 4.7), whereas guanfacine does not. Moreover, imidazoline I2 receptors (I2Rs) are involved in medetomidine-induced impairment of long term potentiation (LTP: a mechanism underpinning learning and memory) in the hippocampus (Takamatsu et al. 2008). This could explain why, at a high dose (10 mg/kg), medetomidine actually impaired memory in wildtypes. The hypothesis that imidazoline

receptors play a role in spatial memory merits further investigation.

Although the NK1R-/- mice tested here did not display any deficits in spatial working memory, the genotype difference in response to 2-AR agonists suggests that there is some interaction between NK1R and 2-ARs, which affects spatial memory. As discussed in Chapter 3,

antagonism or functional ablation of NK1R blunts GABAergic inhibition of locus coeruleus neuronal firing (Maubach et al. 2002; Ebner and Singewald 2007). Moreover, NK1Rs are located on noradrenergic neurones of the LC itself (Chen et al. 2000; Santarelli et al. 2001). This could explain why systemic injection of the NK1R antagonists, WIN 51708 and CP 96345, prevents a clonidine-induced reduction in spiking of LC neurones in rats (Blier et al. 2004). However, if NK1R antagonism reduces the response to 2-AR agonists, it might be expected that mice with functional ablation of NK1Rs are less sensitive to 2-AR agonists. This is certainly the case for medetomidine: this drug had no effect on the spatial memory of NK1R-/- mice, but had clear effects in wildtypes. However, this was not the case for guanfacine. This leads to the inference that there is something exceptional about guanfacine, and perhaps by extension, the 2A-AR subtype, in NK1R-/- mice. Indeed, Fisher et al reported a 70% reduction in [35S]GTPgammaS binding to 2A-ARs in the locus coeruleus of NK1R-/- mice (Fisher et al. 2007). Although the mechanism by which guanfacine improved spatial memory in NK1R-/- was not investigated here, this genotype difference in 2A-ARs could merit further investigation.

Studies of working memory in the most well established model of ADHD, the Spontaneously Hypertensive Rat (SHR), have given inconsistent results (Sontag et al. 2013). Mook et al found that SHRs had better working memory in a radial arm maze, whereas Wyss et al demonstrated 12 month old (i.e. aged) SHRs had impaired memory in the same test compared to control strains (Wyss et al. 1992; Mook et al. 1993).

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122 The results presented here are particularly interesting given reports showing that children and adults with ADHD have deficits in verbal and spatial working memory (Hervey et al. 2004; Martinussen et al. 2005; Vance et al. 2013). This executive dysfunction may form the basis of what is described in diagnosis as ‘inattention’, or indeed vice versa: spatial working memory could depend on selective attention. One functional MRI study suggested the latter could be true: Postle et al (2006) argue that spatial working memory is dependent on selective spatial attention, rather than recall of information.

The results presented in this Chapter suggest that guanfacine could be a better treatment option than non-subtype selective 2-AR agonists, for improving spatial memory/attention, particularly in a subset of ADHD patients carrying the TACR1 gene polymorphism(s).

4.5 Highlights

 NK1R-/- mice display an anxiogenic phenotype compared to wildtypes on a mixed 129/Sv/C57Bl6 x MF1 background strain. This could be because of an interaction between background strain and loss of functional NK1R.

 NK1R-/- mice do not show recognition or spatial memory deficits in tests of short-term memory.

 Guanfacine, at a dose to which NK1R-/- mice are more sensitive (reported in Chapter 3), has no effect on anxiety-like behaviour of NK1R-/- or wildtype mice.

 The same dose of guanfacine improved short-term spatial memory in NK1R-/- mice, only, in an object recognition task. This could help explain guanfacine’s efficacy in treating cognitive abnormalities (particularly inattention) in ADHD.

 Medetomidine also induced a genotype difference in spatial memory, but had no effect on NK1R-/- mice. The difference between the two 2-AR agonists is likely due to subtype selectivity, or differential actions at imidazoline receptors.

123

Chapter 5

The effects of atomoxetine on hyperactivity,

inattention and impulsivityin NK1R-/- and

Chapter 5

124

Chapter 5. The effects of atomoxetine on

hyperactivity, inattention and impulsivity in

NK1R-/- and wildtype mice

5.1 Introduction