Introduction
The cerebellum has long been associated with motor control (Holmes
1917) but has also become recognized recently for its participation in cognitive, language and spatial processing (Strick et al.
2009; D’Mello and Stoodley
2015). This structure is extensively innervated by noradrenergic (NA) axons arising from the locus coeruleus (LC), which consists of neurons that become most active when an individual is in a state of vigilance or arousal (Berridge and Waterhouse
2003; Loughlin et al.
1986; Olson and Fuxe
1971; Sara and Bouret
2012; Pickel et al.
1974; Bloom et al.
1971; Mugnaini and Dahl
1975; Hokfelt and Fuxe
1969; Chandler et al.
2013; Aston-Jones et al.
1991). The significance of cerebellar norepinephrine neurotransmitter in motoric control is highlighted by the finding that depletion of cerebellar norepinephrine impairs acquisition of novel locomotive tasks, such as to run on a rod runway (Watson and McElligott
1984), while the decline of the cerebellar norepinephrine system with aging has been shown to retard the rate of acquisition of motor skills (Bickford
1993; Bickford et al.
1992). This age-related decline is ascribed to changes in the density of β2-adrenergic receptors within the cerebellum. Associative motor learning involving the cerebellum, such as delay eyeblink conditioning, also depends on the cerebellar NA system, since norepinephrine is released in the cerebellum precisely during this task and blockade of β-adrenergic receptors in the cerebellum impairs consolidation of this task (Paredes et al.
2009).
Previous work demonstrated that exercise causes an increase in Purkinje cell dendritic field area and total branch length (Pysh and Weiss
1979). Similar to postsynaptic elements including dendritic spines, presynaptic axons of the cerebellar parallel fibers show dynamic structural changes (Carrillo et al.
2013). However, whether exercise evokes structural changes to NA fibers in the cerebellum is a question never before posed but seemed possible, since exercise has already been shown to augment the level of norepinephrine in the locus coeruleus (Dishman
1997) and to evoke changes in norepinephrine metabolism at other terminal fields of LC—the hippocampus and amygdala (Dishman et al.
2000). These exercise-evoked changes have been suggested to confer protection from stress-induced depletion of norepinephrine in the terminal fields (Dishman et al.
2000). There is immense literature indicating that exercise improves learning, memory and cognitive flexibility (for example, (Barrientos et al.
2011; van Praag et al.
2005; Vaynman et al.
2004; Brockett et al.
2015)) as well as resilience to stress (Schoenfeld et al.
2013) through changes in neurotransmitter systems other than the NA system or brain regions other than the cerebellum. We sought to provide complementary data by determining whether mild versus excessive voluntary wheel running activity (WRA) evoked different types of structural plasticity to the NA fibers in the cerebellum.
Neurons of the LC innervate the granule cell layer, the Purkinje cell layer and the molecular layer (Mugnaini and Dahl
1975). We chose to focus our analysis on the molecular layer of the cerebellar cortex, which houses large Purkinje cell (PC) dendritic arbors, because this layer contains the highest level of β-adrenergic receptor-like immunoreactivity within the cerebellar cortex (Aoki et al.
1987). To analyze NA fibers’ structural plasticity following vigorous exercise, we used a paradigm, called activity-based anorexia (ABA). ABA has been used widely for identifying the neurobiological consequences of voluntary excessive exercise (EEX, averaging 15 km/day for rats) that is evoked by temporarily housing animals in an environment with ad libitum access to a running wheel but limited access to food (1 h/day for rats) (Aoki et al.
2012). Fifty years ago, it was shown that when food restriction is combined with wheel access, the majority of the mildly running rats convert to becoming excessive runners, choosing to run, even during the periods of food availability, thereby “self-starving” to death, unless removed from this ABA-inducing environment (Gutierrez
2013; Wable et al.
2015; Routtenberg and Kuznesof
1967). There are a number of theories regarding the reason that food-restricted animals paradoxically become excessive wheel runners, with one being that an innate foraging-like behavior is triggered when starved (Guisinger
2003; Gutierrez
2013). Because hyperactivity, self-starvation and severe weight loss of ABA animals capture the core symptoms of anorexia nervosa, ABA has been used to understand the biological changes evoked within the brain and the body of individuals with anorexia nervosa (Beumont et al.
1994; Casper et al.
2008; Davis et al.
1997). In order to induce mild exercise (EX, ~5 km/day), a separate group of animals had unlimited access to both food and a running wheel. To differentiate the effect of excessive exercise from that of food restriction, cerebellar samples from the EEX and EX groups of rats were compared to samples from age-matched groups of sedentary but food-restricted (FR) rats, and from a control (CON) group of rats that were housed concurrently with ad libitum access to food and no access to a running wheel. Anorexia nervosa is nine times more prevalent in females than in males and emerges during adolescence (ages 14-19) (Kaye
2009). Therefore, we chose to examine the neuroanatomical changes evoked by ABA induction within brains of rats that matched this profile—i.e., adolescent females.
We conducted NA fiber analysis across two sub-divisions of the cerebellum—the D zone of the hemisphere and the A zone vermal modules, because they are functionally distinct (Voogd
2014; Apps and Hawkes
2009; Cerminara and Apps
2011; Apps and Garwicz
2005). For example, the A zone vermal module is composed of alternating zebrin-positive and -negative subzones, each of which receive afferent information from particular regions of the medial accessory olive (Sugihara and Shinoda
2004; Voogd
2014). In contrast, the D zone of the hemisphere has a less organized zebrin pattern, with borders between the subzones that are not easily determined. Moreover, climbing fiber afferents and efferent projections of the hemisphere are distinct from those of the A zone vermal module (Voogd
2014). Their functional subdivisions are dictated by precise inputs onto cerebellar Purkinje cells which, in turn, are the sole output cells of the cerebellum. Thus, the Purkinje cells serve as the nodes for integrating the cerebellum with motor as well as non-motor circuits at all levels (Strick et al.
2009; Sillitoe et al.
2009; D’Mello and Stoodley
2015). Specifically, the longitudinal zones of the cerebellar hemisphere receive little to no input from the spinal cord and are, instead, heavily interconnected to non-motor areas, including the prefrontal cortex that mediates behavioral flexibility (Strick et al.
2009; Stoodley
2015; Balsters et al.
2010). In contrast, the vermal region modules are densely connected with the motor cortex and receive heavy direct innervations from the spinal cord (Coffman et al.
2011; Provini et al.
1968; Strata et al.
2012; Sengul et al.
2014; Reeber et al.
2013). While much is known about the neuronal wiring, less is known about the modules’ contrasting roles in behavior. To date, comparisons of the NA innervation pattern between the vermis and the hemisphere has not been made. Therefore, we also determined whether the NA axonal pattern is different between the two regions in the basal state and whether exercise and/or food restriction evoke changes in the NA innervation pattern differently across the two regions.
We integrated confocal microscopy and Neurolucida digital tracing software to facilitate the acquisition and extraction of structural information about varicosity and NA axons immuno-labeled for dopamine β-hydroxylase (DβH), a marker of norepinephrine-synthesizing neurons and their processes. This approach enabled three-dimensional (3D) reconstruction of spatially distributed NA axonal varicosities from image stacks. We determined voluntary activity-evoked plasticity of NA varicosities, reflecting altered local and global transmission in the molecular layer of the vermis and the hemisphere of cerebellar cortex. Using Voronoi tessellation, we investigated the spatial distribution of the population of varicosities in both regions of interest. Correlation analyses were conducted to identify associations between NA structural plasticity and WRA of individual rats.
Our results suggest distinct modes of operation for LC–NA axons projecting to the cerebellar vermis and hemisphere regions in response to WRA of different intensities, with implications for their differential roles in reducing vulnerability of individuals to ABA.
Acknowledgments
The authors thank Alisa Liu, Clive Miranda and J-Y Wang who assisted with collecting wheel and weight data on the animals. We thank Kei Tateyama for her assistance with analysis of behavioral data. We thank Jan Voogd for encouraging this work and providing literature on the topic, Mario Negrello for discussions of Voronoi tessellation, MBF Bioscience for the Auto-Neuron trial and continuous support. Common Resources at OIST provided access to the confocal microscope. We thank Sho Aoki and Tom Ruigrok for critical discussions, as well as Christopher Yeo and Izumi Sugihara for their input and support. We thank Andy Liu for technical support and Bernd Kuhn for comments on image digitization using the PMT function. We also thank all the researchers who stopped by our SfN poster, providing important comments. This work was part of HN’s dissertation under Marie Curie CEREBNET Project No: 238686 and further supported by Marie Curie Alumni Association (MCAA) Micro one World Grant, The Klarman Foundation Grant Program in Eating Disorders Research to CA, National Institutes for Health grants R21MH091445-01, R21MH105846, R01NS066019-01A1, R25GM097634-01 and R01NS047557-07A1 to CA, NEI Core grant EY13079 to the Center for Neural Science at NYU, UL1 TR000038 from the National Center for the Advancement of Translational Science (NCATS) to TGC, NYU’s Research Challenge Fund to CA, NYU Dean’s Undergraduate Research Fund to Alisa Liu l and J-Y Wang, NYU Abu Dhabi fund to CM, and the JSPS PE13033 to HN and GA.