Spinal Cord Connections

Group Members

Brett Graham – Group Leader
Brett graduated with his PhD in 2006 and after a short postdoctoral period started his research laboratory, now the Spinal Cord Connections Group, in 2008. The primary theme of his research is spinal sensory coding, a topic he has been focussed on since completing Honours year studying inhibitory synaptic transmission between spinal dorsal horn neurons in 2001.

email: brett.graham@newcastle.edu.au
profile: http://www.newcastle.edu.au/profile/brett-graham
ph:02 49215397

Sally Dickinson – Research Assistant
Sally graduated with a B.Biomed Sci degree in 2013 and joined the Spinal Cord Connections Group soon after. She supports a range of projects, recovering the morphology of neuobiotin filled cells, fixed tissue immunocytochemistry, and mRNA expression analysis.

email: sally.dickinson@newcastle.edu.au
ph:02 49854282

Jamie Flynn – PhD Candidate
Jamie first undertook a summer scholarship with the Spinal Cord Connections Group between the 2nd and 3rd years of his degree. Then, after completing his B.Biomed Sci degree he joined the Additicon Neurobiology Group (Chris Dayas) and worked as a research assistant for 12 months. He rejoined our Group in 2009/10 and completed an Honours project developing a retrograde tracing preparation to study the electrophysiological properties of a select group of long propriospinal neurons that connect the cervical and lumbar spinal regions. He achieved a grade of First Class for his work and then continued these experiments in his current role as a PhD candidate. His studies have continued to analyse propriospinal neurons under normal and pathological conditions (spinal cord injury). He has also undertaken a series of studies in Prof Martyn Goudling’s Laboratory (Salk Institute, US) to analyse the neurotransmitter phenotype and developmental genetics of this population.

email: jamie.flynn@newcastle.edu.au
ph:02 49854282

Mark Gradwell – PhD Candidate
Mark graduated with a B.Biomed Sci Hons degree in 2013 and then went on to work as a Research Assistant in the first half of 2014, developing a spinal cord and attached gut preparation for patch clamp analysis of visceral processing. He then joined the Spinal Cord Connections Group in late 2014 as a PhD candidate. His project aims to understand the role of a particular type of inhibitory interneuron in the dorsal horn that expresses parvalbumin (PV). Our group has previously shown that PV neurons make axoaxonic connections with primary afferents and Mark is now exploring the functional relevance of these connections.

email: mark.gradwell@uon.edu.au
ph:02 49854282

Jessica Madden – Research Assistant
Jess first undertook a 3rd year undergraduate project in the Spinal cord Connections Group in 2010 looking at the impact of arthritis on spinal sensory coding. She went on to work as a Research Assistant in the Group before completing an Honours project in 2013/14 studying electrophysiology,gene expression, and neurochemical phenotype of propriocecptive dorsal root ganglion neurons labeled with green fluorescent protein. After completing her project and being awarded a grade of First Class she has conintued working with the group as a research assistant.

email: jessica.madden@newcastle.edu.au
ph:02 49854282

Kelly Smith – PhD Candidate
Kelly first undertook a 3rd year undergraduate project in the Spinal cord Connections Group in 2010 looking at the impact of arthritis on spinal sensory coding. She went on to complete an Honours project in 2011 assessing the role of pro and anti-inflammatory cytokines in regulating sensory processing circuits in the spinal dorsal horn, achieving a grade of First Class. In 2012/13 Kelly travelled abroad, with a strong dose of the ‘travel bug’ but then rejoined the Spinal Cord Connections Group in the second half of 2013 as a PhD candidate. Her project aims to understand the role of excitatory interneurons in dorsal horn sensory processing circuits, focussing on a particular group of neurons that expresses calretinin (CR). Kelly is using patch clamp electrophysiology coupled with optogenetics to study the properties and connections of these neurons under normal and pathological conditions.

email: ksmith5@uon.edu.au
ph:02 49854282



The spinal cord plays a similar role to a telephone exchange

Research Focus
The spinal cord is much like a telephone exchange, receiving information from a multitude of channels, which must be preserved and processed before they can be directed to appropriate destinations. We know that in spinal cord injury those lines of communication are severed, halting the transmission of vital information and causing a loss of sensation and movement below the injury site. In chronic pain, these communication lines can become crossed and information is redirected to inappropriate destinations with the potential to make a gentle touch cause excruciating pain. Similarly, many movement disorders can be likened to a situation where communication lines are either crossed or broken with the consequence being a loss of smooth, efficient, coordinated movement.

In our efforts to understand and treat this range of spinally-based conditions, knowledge about how the lines of communication in this region are connected normally is critical if we are to repair and rewire them after damage. This is a task that has long been considered too immense given the sheer number of different nerve cell types that are interconnected in spinal cord networks, and the lack of anatomical organisation – ie, unlike a telephone exchange where wires and cables are organised in a roughly ordered manner, the connections of the spinal cord are intermingled in a chaotic and disorganised mosaic. Fortunately, a number of recent scientific breakthroughs have now given us tools to understand how spinal networks are connected and disconnected by disease and injury.


The recovered morphology of a calretinin positive dorsal horn neuron. The white processes are dendrites, which receive information from other populations of neurons. The red processes are the axon, which conveys information from the pictured neuron to other nerve cell populations.


LASU imageOur laboratory includes a brain tissue slicing station, two state of the art in vitro electrophysiology setups, a dedicated laser stimulating and uncaging (LASU) setup, an in vivo electrophysiology setup, and a behavioural testing facility for assessing sensory thresholds and pain in rodents.  Experiments span from single channel analysis of individual receptor properties, to synaptic and intrinsic membrane properties of neurons using in vitro CNS slice preparations, and also whole animal in vivo studies of single neuron properties, and responses to natural stimuli.


1. The role of Hypothalamic Orexin circuits in neuropathic pain
Background: The basis for interactions between chronic pain and other mood-related conditions is poorly understood. This project seeks to understand the role of a brain region known as the lateral hypothalamus in chronic pain signalling. Studies will focus on a specific population of LH neurons that express the neuropeptide orexin. Importantly, orexin has been shown to have analgesic effects but is also strongly implicated in regulating sleep, feeding, reward seeking, depression, and anxiety.
Animals: Orexin eGFP mice
Approach: Experiments will compare sham operated orexin eGFP mice with a group that have undergone the spared nerve injury model of neuropathic pain. The properties of orexin eGFP positive neurons will then be characterised in hypothalamic brain slices from each animal group.
1. Orexin eGFP mice will undergo sensory threshold testing using von Frey filaments to determine their baseline mechanical sensitivity. One group of animals will then undergo spared nerve injury surgery that has previously been shown to alter mechanical thresholds and replicate neuropathic pain-like symptoms. Another group will undergo sham surgery without nerve injury.
2. Mice will then recover from surgery and sensory threshold testing repeated on days 3, 5, and 7 after surgery to monitor changes in mechanical sensitivity.
3. After day 7 testing, hypothalamic brain slices will be prepared and patch clamp recordings obtained from orexin eGFP-positive lateral hypothalamic neurons to characterise their intrinsic excitability and synaptic inputs.
2. The effect of ageing on spinal pain circuits
Background: A substantial body of epidemiologic work suggests that pain is most common during the late middle-aged phase of life (55-65 years) and that this elevated incidence continues into older age (65+). Furthermore, these statistics remain broadly true regardless of the anatomical site or the pathogenic cause of pain. Preclinical, animal-based work on the relationship between ageing and pain is less clear with some work suggesting that thermal and mechanical reflex thresholds become more sensitive, others report decreased sensitivity, and some finding no difference between young and old animals. Despite this confusion, a recent review indicates that increased sensitivity is most common, in agreement with the clinical data. The search for a biological basis to explain changes in pain perception throughout lifespan has provided a number of possible substrates. For example, neurons in the spinal cord of aged animals exhibit enhanced excitability with 1) enhanced spontaneous action potential (AP) discharge, 2) elevated AP discharge during noxious thermal stimulation of the hindpaw, and 3) expanded high threshold receptive fields.
Animals: 3-6 and 24-30 month old C57Bl/6 mice
Approach: Experiments will using and in vitro spinal cord slice preparation to compare sensory processing circuits in young (3-6 months) and old (24-30 months) adult mice.
Experiments will broadly follow our previous studies providing and electrophysiological, morphological and neurochemical analysis of dorsal horn neurons in spinal cord slices from young and old mice. This will include patch-clamp recordings to assess the intrinsic excitability, synaptic transmission, and primary afferent input using dorsal root stimulation. Experiments will also assess circuit level behaviour using pharmacological approaches to induce epileptiform activity. Finally, recoded neurons will be recovered via neurobiotin-filling and stained for a range of established neurochemical markers.
3. Molecular/genetic analysis of spinal circuits
Background: The spinal dorsal horn contains a heterogenous population of neurons that mediate spinal sensory coding. Surprisingly our knowledge of discrete neuronal types within these circuits and their precise role in sensory processing is extremely limited. Until recently we have been forced to collect data from multiple (unidentified) neuronal classes and then ‘pool’ these results to provide an ‘averaged view’ of sensory function (or dysfunction). This approach clearly overlooks the diversity of cell types in the DH originally described more than a century ago by Ramon y Cajal (1899). Since Cajal’s work there has been general agreement that if we are to understand nervous system function we must first understand how various neuron types are assembled into processing circuits. This application aims to address these deficits in our understanding by building a more complex picture of dorsal horn sensory processing.
Animals: Calretinin eGFP mice, Parvalbumin eGFP mice, Thy1 eGFP mice
Approach: A molecular screening approach will be used to dissect neuronal heterogeneity. In this approach the gene expression profile of individual, or small groups of neurons (identified via GFP expression) will be characterized using single-cell quantitative PCR (qPCR) analysis. These data can then compared across samples to identify subsets of cells with similar profiles. This establishes subpopulations that can be considered distinct according to molecular criteria, information that can then be used to predict the function of different subpopulations as well as providing novel electrophysiological and anatomical signatures.
1. The spinal cord of calretinin, parvalbumin, and Thy1-eGFP mice will be extracted and sectioned in parasagittal slices. The dorsal horn region of slice will then be microdissescted and pooled for dissociation.
2. Spinal cord slices are dissociated using a light digestion followed by gentle trituration with a series of flame polished pipettes of increasingly smaller tip diameter.The dissociated homogenates are then centrifuged, the tissue debris in the supernatant is removed and neurons are resuspended.
3. Neuron suspensions are plated under a microscope and and a micropipette is used to collect eGFP expressing neurons using gentle suction. Neurons are either collected individually or pooled in the collection pipette.
4. RNA is then collected these neurons reverse transcribed and transcripts of interests are amplified and quantified using real-time PCR
4. Characterisation of Thy1 positive neurons in the dorsal horn
Background: We have undertaken a pilot characterisation of Thy1 positive dorsal horn neuron in a Thy1-eGFP mouse line maintained by Rohan Walker. This data suggests that the Thy1 population are a novel cell type that is usually excluded from analysis. This is because they have an unusual and complex morphology that does not fit into the routinely used classification schemes. As such, this project aims to selectively record from and characterise this population – how they fire APs, synaptic inputs, neurotransmitter and neurochemcial phenotype
Animals: Thy1eGFP mice
Approach: Experiments will broadly follow our previous studies using targeted recordings to characterise specific populations (pavalbumin-positive and calretinin-positive neurons). This will include patch-clamp recording in spinal cord slices and recovery of neurobiotin-filled neurons.
Experiments: Patch clamp recordings from Thy1-eGFP dorsal horn neurons in spinal slices, characterise their intrinsic excitability, synaptic inputs, and primary afferent input using dorsal root stimulation.
5. Characterisation of calretinin positive neurons in the deep dorsal horn of the sacral spinal cord
Background: An unidentified population of neurons in the deep dorsal horn of the spinal cord are labelled in our calretinin-eGFP mice. We have already excluded the possibility that these neurons are preganglionic parasympathetic neurons, which reside in the general region these calretinin-eGFP neurons are found. Another possibility is that these neurons are spinocerebellar. This project seeks to characterise deep dorsal horn calretinin-positive neurons, determine their identity and function.
Animals: Calretinin-EGFP, Calretinin-Channelrhodopsin-2
Approach: Experiments will broadly follow our previous studies using targeted recordings to characterise specific populations (parvalbumin-positive and calretinin-positive neurons) in the superficial dorsal horn. In addition, we will use the calretinin-channelrhodpsin-2 mouse to photostimulate this population and assess their functional role.
1. Brain injection and tracing studies to test if calretinin-positive deep dorsal horn neurons are spinocerebellar. Tracer will be injected in tho the cerebellum and then animals will be sacrificed after a wait period to allow tracer migration. Spinal cords are then processed to assess for colocalisation of tracer and calretinin-eGFP in the deep dorsal horn.
2. Make patch clamp recordings from calretinin-positive deep dorsal horn neurons in spinal slices, characterise their intrinsic excitability, synaptic inputs, and primary afferent input using dorsal root stimulation.
3. Prepare spinal slices and place them on a microelectrode array for multiunit recording. Systematically photostimulate in the deep dorsal horn to activate calretinin-channelrhodopsin-2 neurons and monitor the resulting spread of electrical activity in the slice to determine the associated spinal circuitry.
4. Behavioural analysis of calretinin positive deep dorsal horn neurons using optogenetics to activate/inactivate them. Surgery will be performed to position optic fibre guide canula in the deep dorsal horn of the sacral spinal cord. Animals are then allowed to recover for several days before the canula is connected to to an LED light source, delivering photostimulation to calretinin positive deep dorsal horn neurons that express Channelrhodopsin-2. The behavioural consequence of selectively activating this population will then be assessed using a variety of behavioural paradigms.

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