The role of DA in the control of locomotion

The role of DA in locomotor systems is particularly pertinent, as a loss of dopaminergic neurons underlies the neurodegenerative motor disorder, Parkinson’s disease (for review see Dauer & Przedborski 2003). While the pathophysiology of the disease is beyond the scope of this overview, DA is clearly important for proper motor behaviour since the majority of parkinsonian symptoms involve impaired movements – these include tremor at rest, rigidity, a festinating gait, and compromised voluntary movements. Moreover, the major drug for the treatment of Parkinson’s disease is the DA precursor L-DOPA. In addition to Parkinson’s disease, DA dysfunction is also linked to several other

conditions related to movement including Huntington’s disease (Andre et al, 2010) and restless leg syndrome (Clemens et al. 2006).

The dopaminergic pathway most commonly associated with locomotion, and indeed the disrupted pathway in Parkinson’s disease, is the nigrostriatal pathway. The midbrain dopaminergic populations in the SNc and VTA in mammals project rostrally to the basal ganglia. Classically, there are thought to be two parallel pathways acting in the basal ganglia; namely the direct and indirect pathways (Smith & Kieval 2000).

Activation of the direct pathway is linked to increased movement while activation of the indirect pathway is thought to cause reduced movement. Recently, experimental

evidence has corroborated this theory showing that optogenetic activation of neurons in the indirect pathway impairs movement, while activation of direct pathway neurons facilitates locomotion (Kravitz et al. 2010). Moreover, in a mouse model of Parkinson’s disease, activation of the direct pathway neurons rescued specific deficits in movement.

The basal ganglia are thought to mediate its control on locomotion via descending projections to the MLR and DLR, which in turn activate reticulospinal nuclei in the caudal brainstem. Very recently, an additional dopaminergic pathway has been

described in the lamprey, originating from the PT, an area deemed equivalent to the SNc due to its striatal projections (Ryczko et al. 2013; Pombal et al, 1997b). It provides a direct descending link between the A11 dopaminergic neurons of the PT and the MLR

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and shows that DA released in the MLR activates D1-like DA receptors to activate locomotion. In addition, the A11 population projects directly onto neurons of the spinal cord, and is thought to be the sole source of spinal DA in vertebrates (Skagerberg & Lindvall, 1985; Qu et al, 2006).

In the lamprey, DA has been shown to have a dose-dependent effect on NMDA-induced locomotion (Svensson et al. 2003; McPherson & Kemnitz 1994). Low doses (<10M) of DA increased burst frequency while high doses (>10M) decreased it. Additionally, slow perfusion of 200M DA mimics this effect showing a biphasic response as the drug concentration builds up (Svensson et al, 2003). Furthermore, the effects are found to be due to endogenous DA since bupropion, a DA re-uptake blocker, causes a similar biphasic effect on motor burst frequency.

In zebrafish, DA reduces the occurrence of spontaneous swimming at 3dpf. This effect is mediated via D2-like receptors and is caused by endogenous release of DA in the brain, implicating a supraspinal mode of action (Thirumalai & Cline 2008). The

inhibitory effects of D2 receptor activation are dependent on a reduction in cAMP since artificially increasing cAMP levels with forskolin blocked the effects of DA. By 5dpf, zebrafish display a different pattern of spontaneous swimming; episodes occur more frequently but are shorter in duration. Exogenous DA inhibited swimming, returning the spontaneous locomotor output to that resembling a younger animal. However,

increasing endogenous DA or blockade of D2 receptors no longer altered the motor pattern. This was despite dopaminergic neurons still being present at these stages (McLean & Fetcho 2004), pointing to a transient role for DA during development, which is superseded or masked as the network matures (Thirumalai & Cline, 2008).

DA released in the spinal cord can potentially activate any one of the 5 known receptor subtypes (Zhu et al, 2007). Moreover, a role in locomotion is predicted based on the expression of receptors in the grey matter of the mouse lumbar cord, including post- synaptic expression in MNs. Interestingly there is a predominance of D2 receptors, suggesting a leading role for this subtype in the spinal cord.

131 D1 agonist D1 agonist + D2 antagonist No drug D2 antagonist No drug D2 agonist D2 agonist + D2 antagonist

A

Bi

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Figure 2.6 Activation of D1-like and D2-like DA receptors have opposing effects in the nervous system.

A, In the rat neostriatum the formation of cAMP is dependent on DA receptor sub-type

activation. SKF 38393, a D1-like DA receptor agonist increases cAMP efflux (upper panel) and this effect is further enhanced in the presence of a D2-like receptor antagonist, supiride. On the other hand, LY-141865, a D2-like DA receptor agonist reduces cAMP efflux (lower panel) and

this effect is blocked by application of sulpiride. B, In neurons in the spinal ganglia of the rat

DA mediates differential effects causing depolarisation (Bi), hyperpolarisation (Bii) and mixed

responses where an initial hyperpolarisation is superseded by depolarisation (Biii). The

depolarising effects are predicted to be based on D1-like DA receptor activation leading to elevated cAMP levels and the activation of cAMP-dependent cation channels. The

hyperpolarisations were blocked by sulpiride while depolarisations were only blocked by the non-selective DA receptor antagonist, haloperidol (not shown), supporting the idea that the effects are mediated by differential activation of DA receptor subtypes. Figures adapted from Stoof & Kebabian (1981; A) and Abramets & Samoilovich (1991; B).

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The actions of D1- and D2-like receptors are broadly characterised as excitatory and inhibitory, based on their ability to either increase or decrease cAMP levels,

respectively (Stoof & Kebabian, 1981; Fig. 2.6). This distinction is preserved in the spinal cord. In mice with a spinal transection, D1/D5 agonists, but not D2-like agonists, are able to elicit rhythmic locomotor activity (Lapointe et al, 2009). At the level of the MNs, these effects are mediated by modulation of AMPA receptor currents and D1- but not D2-like agonists potentiate these excitatory currents (Han et al. 2007; Han &

Whelan 2009). Sensory motor pathways are also mediated by dopaminergic signalling within the spinal cord and D2-like receptors are known to mediate depression of the monosynaptic stretch reflex in the cat (Carp & Anderson, 1982), rat (Gajendiran et al, 1996) and mouse,where the effect is mediated by a specific action at D3 receptors (Clemens & Hochman 2004). Additional effects on spinal MNs include a depolarisation and an increase in firing frequency, shifting the slope of f-I plots to the left (Han et al, 2007). These effects are mediated by apamin-sensitive SKCa channels resulting in a

reduction in the post-spike medium afterhyperpolarisation (~100-200ms; mAHP). A similar reduction in AHP is found in lamprey MNs where DA, acting via D2-like receptors, reduces calcium entry following action potentials, thereby preventing KCa

activation (Schotland et al. 1995; McPherson & Kemnitz 1994). Furthermore, DA is predicted to affect low-threshold, fast-inactivating potassium channels since it mimics the effects of the potassium channel antagonist 4-aminopyridine (4-AP). Both DA and 4-AP reduce the latency to the first spike after current injection and are predicted to achieve this via a reduction of an A-type potassium current (IA).

The effects of DA on rhythmic locomotion are not solely based on effects at the level of MNs. Hb9+ interneurons are known to be active during locomotion and are thought to be important for locomotor rhythm generation in the mammalian spinal CPG (Wilson et al. 2005). DA, in combination with 5-HT and NMDA, is necessary but not sufficient to generate rhythmic oscillations in these neurons (Han et al, 2007), highlighting the importance of multiple modulators acting on both MNs and spinal interneurons for the generation of properly coordinated locomotion.

In general, effects of DA on motor rhythm generation have been attributed to D1-like receptor activation, whilst D2-like receptor activation has been thought to mostly mediate the modulation sensory pathways. However, there is good evidence that differential DA receptor activation can occur within the same basic circuitry. In striatal

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neurons, D1-like receptor activation potentiates NMDA-mediated responses, while D2- like receptor activation attenuates them (Cepeda et al. 1993). Locomotion in rats is increased by D1-like receptor activation in the ventral palladium and decreased by D2- like receptor activation (Gong et al. 1999). Locomotion in C.elegans is mediated by opposing actions of DA on the same MNs, acting at either D1-like DOP-1 receptors to increase locomotor speed; or at D2-like DOP-3 receptors to reduce locomotor speed (Chase et al. 2004). Moreover, differential activation of the two opposing receptors promotes either Gq or Go signalling, and subsequently increases or decreases ACh

release from the MNs.

In pro-metamorphic Xenopus tadpoles, DA also displays differential effects on

locomotor output mediated by opposing actions at D1- and D2-like receptors within the spinal cord (Clemens et al. 2012; Fig. 2.7). In this case the actions of DA occur in a dose-dependent fashion: low levels of exogenous DA (2M) exert a net inhibitory effect on spontaneous locomotor activity, reducing the number of motor bursts, swim episodes and the frequency of episodes; while high levels of DA (50M) potentiate locomotor activity, increasing motor bursts, swim episodes and episode frequency. By using a range of DA concentrations, the authors hypothesised that they would preferentially activate high affinity D2-like receptors or low affinity D1-like receptors. This was confirmed by showing agonists at both receptor types mimic the corresponding dose- dependent response (i.e. The D1-like agonist SKF 38393 potentiated locomotor activity, mimicking the effects of 50M DA). Moreover the respective antagonists at each receptor type opposed these effects.

In this chapter I have extended the analysis of DA and NO effects on locomotor activity in pro-metamorphic Xenopus tadpoles and paid particular attention to the likely sites of action and possible interactions between these two modulatory systems.

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Dopamine concentration

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Figure 2.7 Opposing effects of D1- and D2-like DA receptor activation on motor output in pro-metamorphic Xenopus tadpoles.

Ai, DA modulates the occurrence of spontaneous locomotor output in pro-metamorphic

Xenopus tadpoles in a dose-dependent fashion. Low concentrations (2M) of DA cause a

reduction in motor output relative to control while high concentrations (50M) cause an

increase (Ai). These effects are mediated via differential activation of either D1-like or D-2 like

DA receptors. D1 agonists mimic high DA and cause and increase in motor output (Aii) while

D2-like DA receptor agonists mimic low DA and cause a decrease in motor output (Aiii). The

respective antagonists at these receptors have the opposite effect on motor output (Aii & Aiii).

Bi, A schematic of the effects of DA on the spinal CPG for locomotion in Xenopus tadpoles

shows that DA released from supraspinal centres acts in a dose-dependent manner to activate

either DA- or D2-like DA receptors. Bii, The modulatory effects on the motor output are

directly opposite with high DA activating D1-like receptors and causing increased motor output and low DA activating D2-like DA receptors to mediate a reduction in motor output. Figures adapted from Clemens et al, (2012).

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