Jamie R. Flynn

Cocaine- and Amphetamine-Regulated Transcript (CART) Signaling within the Paraventricular Thalamus Modulates Cocaine-Seeking Behaviour

BACKGROUND Cocaine- and amphetamine-regulated transcript (CART) has been demonstrated to play a role in regulating the rewarding and reinforcing effects of various drugs of abuse. A recent study demonstrated that i.c.v. administration of CART negatively modulates reinstatement of alcohol seeking, however, the site(s) of action remains unclear. We investigated the paraventricular thalamus (PVT) as a potential site of relapse-relevant CART signaling, as this region is known to receive dense innervation from CART-containing hypothalamic cells and to project to a number of regions known to be involved in mediating reinstatement, including the nucleus accumbens (NAC), medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). METHODOLOGY/PRINCIPAL FINDINGS Male rats were trained to self-administer cocaine before being extinguished to a set criterion. One day following extinction, animals received intra-PVT infusions of saline, tetrodotoxin (TTX; 2.5 ng), CART (0.625 µg or 2.5 µg) or no injection, followed by a cocaine prime (10 mg/kg, i.p.). Animals were then tested under extinction conditions for one hour. Treatment with either TTX or CART resulted in a significant attenuation of drug-seeking behaviour following cocaine-prime, with the 2.5 µg dose of CART having the greatest effect. This effect was specific to the PVT region, as misplaced injections of both TTX and CART resulted in responding that was identical to controls. CONCLUSIONS/SIGNIFICANCE We show for the first time that CART signaling within the PVT acts to inhibit drug-primed reinstatement of cocaine seeking behaviour, presumably by negatively modulating PVT efferents that are important for drug seeking, including the NAC, mPFC and BLA. In this way, we identify a possible target for future pharmacological interventions designed to suppress drug seeking.

Propensity to ‘relapse’ following exposure to cocaine cues is associated with the recruitment of specific thalamic and epithalamic nuclei

The thalamus is considered an important interface between the ventral striatopallidum and the dorsal striatum, and may therefore contribute to compulsive drug-seeking behaviour. Recent evidence suggests that the paraventricular thalamus (PVT), a dorsal midline thalamic nucleus, and the mediodorsal thalamus (MD) are involved in drug self-administration and respond to drug-associated cues. At present, however, the role of these thalamic regions in mediating cue-induced reinstatement of cocaine-seeking is unclear. Similarly, the habenula complex, part of the epithalamus, has been implicated in nicotine self-administration and cue-induced reinstatement of heroin seeking, but the role of this region in cocaine reinstatement behaviour has received little attention. Rats (n=20) were trained to self-administer cocaine in the presence of discriminative stimuli associated with drug availability (S⁺) or drug non-availability (S⁻). Once a stable level of responding was reached, lever pressing was extinguished. Animals were then tested for reinstatement and sacrificed immediately following the presentation of either the S⁻ or S⁺ discriminative stimuli, and Fos-protein expression was assessed in thalamic and epithalamic regions. Interestingly, significant variation was observed in reinstatement behaviour, allowing a comparison between high-reinstating (HR), low-reinstating (LR) and control animals. Compared with LR animals, HR animals exhibited increased Fos-protein expression in the PVT, intermediodorsal thalamus and the medial and lateral divisions of the habenula. Our data provide evidence that activation of thalamic and epithalamic nuclei is associated with propensity to reinstate to cocaine-seeking elicited by drug-related cues. We also build upon existing data highlighting the importance of the PVT in reinstatement behaviour.

Down-regulated striatal gene expression for synaptic plasticity-associated proteins in addiction and relapse vulnerable animals

Reducing the likelihood of relapse represents one of the greatest obstacles in the successful treatment of cocaine addiction. Dysregulation of the synaptic plasticity processes long-term potentiation (LTP) and long-term depression (LTD) is thought to be associated with protracted relapse risk. To improve our understanding of the molecular mechanisms contributing to relapse vulnerability we trained rats (n=52) to self-administer cocaine and phenotyped animals as relapse-vulnerable or relapse-resilient using procedures adapted from Deroche-Gamonet et al. (Science 2004, 305, 1014-1017). Gene expression analysis, targeted at synaptic plasticity-related genes, revealed significant transcript down-regulation in the ventral and dorsal striatum of relapse-vulnerable animals compared to relapse-resilient controls. This included reduced expression of genes encoding proteins implicated in the dendritic translation of synaptic plasticity-related transcripts, the dynamic regulation and trafficking of ionotropic glutamate receptors important for LTP and LTD, along with neuronal surface receptors that initiate downstream signalling pathways associated with synaptic plasticity. Together, our data are consistent with recent reports of an inability to evoke LTD in the striatum of addiction-vulnerable rats. To our knowledge, this is the first study to demonstrate down-regulated synaptic plasticity-associated gene expression not only in the ventral striatum, where the majority of addiction-related synaptic plasticity studies have been conducted, but also in the dorsal striatum of animals categorized as relapse-vulnerable. As these neural correlates were elucidated using an approach incorporating individual behavioural differences, they potentially provide more relevant insight into addiction and assist the development of novel pharmacotherapies to treat relapse.

PEA-CLARITY: 3D molecular imaging of whole plant organs.

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

Measurement of the Vertebral Canal Dimensions of the Neck of the Rat with a Comparison to the Human

The aim of this study was to determine the dimensions of the vertebral canal in the neck of the rat, because little is known about the morphology of the rats cervical spine. A comparison then was made to the vertebral canal in the neck of the human. In part 1 of this study, we determined the precision of three different methods to measure the vertebral canal. The error (coefficient of variation) in these methods was found to range from 1 to 8%. In part 2, we used a computer‐based system to measure digital images of the vertebra and determined the anterior to posterior and the transverse vertebral canal dimensions in the neck of 19 young adult Sprague‐Dawley rats. The anterior to posterior dimension of the vertebral canal was greatest at the upper cervical (C1–C2) level and progressively decreased in the more caudal segments (C3–T1). The transverse dimension was greatest at the atlas (C1) vertebra and smallest at the axis (C2) vertebra with a steady increase in the transverse dimension with more caudal segments and a maximum transverse dimension at the level of the C6 and C7 vertebra. This study has demonstrated that the vertebral canal in the neck of young adult rats is similar in some regards to that of human. However, there are clear differences between the rat and human. These may be associated with differences in the morphology of the spinal cord or postural differences such as the cervicothoracic lordosis in bipeds compared with that in quadrupeds. Anat Rec, 2007.

Functional changes in deep dorsal horn interneurons following spinal cord injury are enhanced with different durations of exercise training

Exercise training after spinal cord injury (SCI) enhances collateral sprouting from axons near the injury and is thought to promote intraspinal circuit reorganisation that effectively bridges the SCI. The effects of exercise training, and its duration, on interneurons in these de novo intraspinal circuits are poorly understood. In an adult mouse hemisection model of SCI, we used whole‐cell patch‐clamp electrophysiology to examine changes in the intrinsic and synaptic properties of deep dorsal horn interneurons in the vicinity of a SCI in response to the injury, and after 3 and 6 weeks of treadmill exercise training. SCI alone exerted powerful effects on the intrinsic and synaptic properties of interneurons near the lesion. Importantly, synaptic activity, both local and descending, was preferentially enhanced by exercise training, suggesting that exercise promotes synaptic plasticity in spinal cord interneurons that are ideally placed to form new intraspinal circuits after SCI.

Exercise training after spinal cord injury selectively alters synaptic properties in neurons in adult mouse spinal cord.

Following spinal cord injury (SCI), anatomical changes such as axonal sprouting occur within weeks in the vicinity of the injury. Exercise training enhances axon sprouting; however, the exact mechanisms that mediate exercised-induced plasticity are unknown. We studied the effects of exercise training after SCI on the intrinsic and synaptic properties of spinal neurons in the immediate vicinity (<2 segments) of the SCI. Male mice (C57BL/6, 9-10 weeks old) received a spinal hemisection (T10) and after 1 week of recovery, they were randomized to trained (treadmill exercise for 3 weeks) and untrained (no exercise) groups. After 3 weeks, mice were killed and horizontal spinal cord slices (T6-L1, 250 μm thick) were prepared for visually guided whole cell patch clamp recording. Intrinsic properties, including resting membrane potential, input resistance, rheobase current, action potential (AP) threshold and after-hyperpolarization (AHP) amplitude were similar in neurons from trained and untrained mice (n=67 and 70 neurons, respectively). Neurons could be grouped into four categories based on their AP discharge during depolarizing current injection; the proportions of tonic firing, initial bursting, single spiking, and delayed firing neurons were similar in trained and untrained mice. The properties of spontaneous excitatory synaptic currents (sEPSCs) did not differ in trained and untrained animals. In contrast, evoked excitatory synaptic currents recorded after dorsal column stimulation were markedly increased in trained animals (peak amplitude 78.9±17.5 vs. 42.2±6.8 pA; charge 1054±376 vs. 348±75 pA·ms). These data suggest that 3 weeks of treadmill exercise does not affect the intrinsic properties of spinal neurons after SCI; however, excitatory synaptic drive from dorsal column pathways, such as the corticospinal tract, is enhanced.

Electrophysiological characterization of spontaneous recovery in deep dorsal horn interneurons after incomplete spinal cord injury

In the weeks and months following an incomplete spinal cord injury (SCI) significant spontaneous recovery of function occurs in the absence of any applied therapeutic intervention. The anatomical correlates of this spontaneous plasticity are well characterized, however, the functional changes that occur in spinal cord interneurons after injury are poorly understood. Here we use a T10 hemisection model of SCI in adult mice (9-10 wks old) combined with whole-cell patch clamp electrophysiology and a horizontal spinal cord slice preparation to examine changes in intrinsic membrane and synaptic properties of deep dorsal horn (DDH) interneurons. We made these measurements during short-term (4 wks) and long-term (10 wks) spontaneous recovery after SCI. Several important intrinsic membrane properties are altered in the short-term, but recover to values resembling those of uninjured controls in the longer term. AP discharge patterns are reorganized at both short-term and long-term recovery time points. This is matched by reorganization in the expression of voltage-activated potassium and calcium subthreshold-currents that shape AP discharge. Excitatory synaptic inputs onto DDH interneurons are significantly restructured in long-term SCI mice. Plots of sEPSC peak amplitude vs. rise times suggest considerable dendritic expansion or synaptic reorganization occurs especially during long-term recovery from SCI. Connectivity between descending dorsal column pathways and DDH interneurons is reduced in the short-term, but amplified in long-term recovery. Our results suggest considerable plasticity in both intrinsic and synaptic mechanisms occurs spontaneously in DDH interneurons following SCI and takes a minimum of 10 wks after the initial injury to stabilize.

A horizontal slice preparation for examining the functional connectivity of dorsal column fibres in mouse spinal cord

In spinal cord injury (SCI) research, axon regeneration across spinal lesions is most often assessed using anatomical methods. It would be extremely advantageous, however, to examine the functional synaptic connectivity of regenerating fibres, using high-resolution electrophysiological methods. We have therefore developed a mouse horizontal spinal cord slice preparation that permits detailed analysis of evoked dorsal column (DCol) synaptic inputs on spinal neurons, using whole-cell patch clamp electrophysiology. This preparation allows us to characterise postsynaptic currents and potentials in response to electrical stimulation of DCol fibres, along with the intrinsic properties of spinal neurons. In addition, we demonstrate that low magnification calcium imaging can be used effectively to survey the spread of excitation from DCol stimulation in horizontal slices. This preparation is a potentially valuable tool for SCI research where confirmation of regenerated, functional synapses across a spinal lesion is critical.

Anatomical and Molecular Properties of Long Descending Propriospinal Neurons in Mice.

Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in GlyT2GFP and GAD67GFP mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI.