An Emerging Role for the Mammalian Target of Rapamycin in “Pathological” Protein Translation: Relevance to Cocaine Addiction

Complex neuroadaptations within key nodes of the brain’s “reward circuitry” are thought to underpin long-term vulnerability to relapse. A more comprehensive understanding of the molecular and cellular signaling events that subserve relapse vulnerability may lead to pharmacological treatments that could improve treatment outcomes for psychostimulant-addicted individuals. Recent advances in this regard include findings that drug-induced perturbations to neurotrophin, metabotropic glutamate receptor, and dopamine receptor signaling pathways perpetuate plasticity impairments at excitatory glutamatergic synapses on ventral tegmental area and nucleus accumbens neurons. In the context of addiction, much previous work, in terms of downstream effectors to these receptor systems, has centered on the extracellular-regulated MAP kinase signaling pathway. The purpose of the present review is to highlight the evidence of an emerging role for another downstream effector of these addiction-relevant receptor systems – the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 functions to regulate synaptic protein translation and is a potential critical link in our understanding of the neurobiological processes that drive addiction and relapse behavior. The precise cellular and molecular changes that are regulated by mTORC1 and contribute to relapse vulnerability are only just coming to light. Therefore, we aim to highlight evidence that mTORC1 signaling may be dysregulated by drug exposure and that these changes may contribute to aberrant translation of synaptic proteins that appear critical to increased relapse vulnerability, including AMPARs. The importance of understanding the role of this signaling pathway in the development of addiction vulnerability is underscored by the fact that the mTORC1 inhibitor rapamycin reduces drug-seeking in pre-clinical models and preliminary evidence indicating that rapamycin suppresses drug craving in humans

Effects of maternal separation on brain stress systems: Modulation by voluntary exercise in male rats

Early life stress (ELS) has been shown to predispose animals to anxiety- and depression-like behaviour in adulthood. Recent evidence suggests that repeated stress in adulthood dysregulates the hypothalamic orexin/hypocretin system. The current study examined the effects of maternal separation (MS), a well validated rodent model of ELS, on the expression of anxiety-like behaviour following the re-exposure to stress in adulthood. The pattern of Fos-expression in hypothalamic orexin neurons and stress sensitive brain regions was also characterised. Finally, this study examined whether the effects of this double-hit of stress could be reversed using a voluntary exercise intervention during early adulthood. Male rat pups (n=25) were removed from dams for 3hrs on postnatal days (PND) 2-14 (MS). Controls (C; n=25) remained undisturbed during this period except for weekly weighing. On PND 75, animals were randomly allocated to either a ‘stress’ (30min restraint stress) or ‘no stress’ condition (S or NS). A subset of MS animals (n=6) was allowed access to exercise wheels for 1hr/day from PND 40-70. Following this, all animals were behaviourally tested in the open field apparatus for 10mins. Two hours after initiation of restraint, animals were perfused and brains were processed for Fos-protein immunohistochemistry and co-labelled for orexin or tyrosine-hydroxylase (TH). Counts of Fos-positive neurons were made in the hypothalamus, paraventricular nucleus (PVN), paraventricular thalamus (PVT) and ventral tegmental area (VTA). MS-NS rats exhibited behaviour that was indistinguishable from C-NS rats. However, male MS-S rats exhibited decreased exploratory behaviour in the open field task compared to C-S rats. This was associated with a decrease in the percentage of Fos-positive orexin cells in the hypothalamus and reduced Fos-protein in the PVN, PVT and TH-positive VTA cells compared to C-S rats. Interestingly, the exercise intervention reversed the behavioural effects of MS following stress and normalized orexin cell and VTA-TH cell Fos-expression. In conclusion, MS resulted in altered open field behaviour and hypoactivation of the orexin system in response to adult stress. The current study indicates that changes in orexin system function may involve altered activity in stress-sensitive brain regions such as the VTA, PVN and PVT. Importantly, the behavioural and neural changes observed were reversed by voluntary exercise in early adulthood. These findings highlight the importance of non-pharmacological interventions in the treatment of stress-related disorders

Recruitment of hypothalamic orexin neurons after formalin injections in adult male rats exposed to a neonatal immune challenge

Exposure to early life physiological stressors, such as infection, is thought to contribute to the onset of psychopathology in adulthood. In animal models, injections of the bacterial immune challenge, lipopolysaccharide (LPS), during the neonatal period has been shown to alter both neuroendocrine function and behavioural pain responses in adulthood. Interestingly, recent evidence suggests a role for the lateral hypothalamic peptide orexin in stress and nociceptive processing. However, whether neonatal LPS exposure affects the reactivity of the orexin system to formalin-induced inflammatory pain in later life remains to be determined. Male Wistar rats (n=13) were exposed to either LPS or saline (0.05mg/kg, i.p) on postnatal days (PND) 3 and 5. On PND 80-97, all rats were exposed to a subcutaneous hindpaw injection of 2.25% formalin. Following behavioural testing, animals were perfused and brains processed for Fos-protein and orexin immunohistochemistry. Rats treated with LPS during the neonatal period exhibited decreased licking behaviours during the interphase of the formalin test, the period typically associated with the active inhibition of pain, and increased grooming responses to formalin in adulthood. Interestingly, these behavioural changes were accompanied by an increase in the percentage of Fos-positive orexin cells in the dorsomedial and perifornical hypothalamus in LPS-exposed animals. Similar increases in Fos-protein were also observed in stress and pain sensitive brain regions that receive orexinergic inputs. These findings highlight a potential role for orexin in the behavioural responses to pain and provide further evidence that early life stress can prime the circuitry responsible for these responses in adulthood

Transgenic cross-referencing of inhibitory and excitatory interneuron populations to dissect neuronal heterogeneity in the dorsal horn

The superficial dorsal horn (SDH, LI-II) of the spinal cord receives and processes multimodal sensory information from skin, muscle, joints and viscera then relays it to the brain. Neurons within the SDH fall into two broad categories, projection neurons and interneurons. The later can be further subdivided into excitatory and inhibitory types. Traditionally, interneurons within the SDH have been divided into overlapping groups according to their neurochemical, morphological and electrophysiological properties. Recent clustering analyses, based on molecular transcript profiles of cells and nuclei, have predicted many more functional groups of interneuron than expected using traditional approaches. In this study, we used electrophysiological and morphological data obtained from genetically-identified excitatory (vGLUT2) and inhibitory (vGAT) interneurons in transgenic mice to cluster them into groups sharing common characteristics, and subsequently determined how many clusters will be assigned by combinations of these properties. Consistent with previous reports, we show differences exist between excitatory and inhibitory interneurons in terms of their excitability, nature of ongoing excitatory drive, action potential properties, sub-threshold current kinetics, and morphology. The resulting clusters based on statistical and unbiased assortment of these data fell well short of the numbers of molecularly predicted clusters. There was no clear characteristic that in isolation defined a population, rather multiple variables were needed to predict cluster membership. Importantly though, our analysis highlighted the appropriateness of using transgenic lines as tools to functionally subdivide both excitatory and inhibitory interneuron populations

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

Projection neuron axon collaterals in the dorsal horn: placing a new player in spinal cord pain processing

The pain experience depends on the relay of nociceptive signals from the spinal cord dorsal horn to higher brain centres. This function is ultimately achieved by the output of a small population of highly specialised neurons called projection neurons (PNs). Like output neurons in other CNS regions, PNs are invested with a substantial axon collateral system that ramifies extensively within local circuits. These axon collaterals are widely distributed within and between spinal cord segments. Anatomical data on PN axon collaterals has existed since the time of Cajal, however, their function in spinal pain signalling remains unclear and is absent from current models of spinal pain processing. Despite these omissions, some insight on the potential role of PN axon collaterals can be drawn from axon collateral systems of principal or output neurons in other CNS regions such as the hippocampus, amygdala, olfactory cortex and ventral horn of the spinal cord. The connectivity and actions of axon collaterals in these systems have been well defined and used to confirm crucial roles in memory, fear, olfaction and movement control, respectively. We review this information here and propose a framework for characterising PN axon collateral function in the dorsal horn. We highlight that experimental approaches traditionally used to delineate axon collateral function in other CNS regions are not easily applied to PNs because of their scarcity relative to spinal interneurons, and the lack of cellular organisation in the dorsal horn. Finally, we emphasise how the rapid development of techniques such as viral expression of optogenetic or chemogenetic probes can overcome these challenges and allow characterisation of PN axon collateral function. Obtaining detailed information of this type is a necessary first step for incorporation of PN collateral system function into models of spinal sensory processing

Orexinergic Input to Dopaminergic Neurons of the Human Ventral Tegmental Area

The mesolimbic reward pathway arising from dopaminergic (DA) neurons of the ventral tegmental area (VTA) has been strongly implicated in reward processing and drug abuse. In rodents, behaviors associated with this projection are profoundly influenced by an orexinergic input from the lateral hypothalamus to the VTA. Because the existence and significance of an analogous orexigenic regulatory mechanism acting in the human VTA have been elusive, here we addressed the possibility that orexinergic neurons provide direct input to DA neurons of the human VTA. Dual-label immunohistochemistry was used and orexinergic projections to the VTA and to DA neurons of the neighboring substantia nigra (SN) were analyzed comparatively in adult male humans and rats. Orexin B-immunoreactive (IR) axons apposed to tyrosine hydroxylase (TH)-IR DA and to non-DA neurons were scarce in the VTA and SN of both species. In the VTA, 15.062.8% of TH-IR perikarya in humans and 3.260.3% in rats received orexin B-IR afferent contacts. On average, 0.2460.05 and 0.0560.005 orexinergic appositions per TH-IR perikaryon were detected in humans and rats, respectively. The majority(86–88%) of randomly encountered orexinergic contacts targeted the dendritic compartment of DA neurons. Finally, DA neurons of the SN also received orexinergic innervation in both species. Based on the observation of five times heavierorexinergic input to TH-IR neurons of the human, compared with the rat, VTA, we propose that orexinergic mechanism acting in the VTA may play just as important roles in reward processing and drug abuse in humans, as already established well in rodents

Spinoparabrachial projection neurons form distinct classes in the mouse dorsal horn

Projection neurons in the spinal dorsal horn relay sensory information to higher brain centres. The activation of these populations is shaped by afferent input from the periphery, descending input from the brain, and input from local interneuron circuits. Much of our recent understanding of dorsal horn circuitry comes from studies in transgenic mice; however, information on projection neurons is still based largely on studies in monkey, cat, and rat. We used viral labelling to identify and record from mouse parabrachial nucleus (PBN) projecting neurons located in the dorsal horn of spinal cord slices. Overall, mouse lamina I spinoparabrachial projection neurons (SPBNs) exhibit many electrophysiological and morphological features that overlap with rat. Unbiased cluster analysis distinguished 4 distinct subpopulations of lamina I SPBNs, based on their electrophysiological properties that may underlie different sensory signalling features in each group. We also provide novel information on SPBNs in the deeper lamina (III-V), which have not been previously studied by patch clamp analysis. These neurons exhibited higher action potential discharge frequencies and received weaker excitatory synaptic input than lamina I SPBNs, suggesting this deeper population produces different sensory codes destined for the PBN. Mouse SPBNs from both regions (laminae I and III-V) were often seen to give off local axon collaterals, and we provide neuroanatomical evidence they contribute to excitatory input to dorsal horn circuits. These data provide novel information to implicate excitatory input from parabrachial projection neuron in dorsal horn circuit activity during processing of nociceptive information, as well as defining deep dorsal horn projection neurons that provide an alternative route by which sensory information can reach the PBN

Neurokinin 1 receptor blockade in the medial amygdala attenuates alcohol drinking in rats with innate anxiety but not in Wistar rats

Background and Purpose: Substance P and its preferred neurokinin receptor NK1 have been implicated in stress and anxiety and have been proposed as possible therapeutic targets for the treatment of anxiety/depression. Attention is also being focused on the role this neuropeptide system may play in drug addiction, because stress-related mechanisms promote drug abuse. Experimental Approach: The effects of the rat-specific NK1 receptor antagonist, L822429, on alcohol intake and seeking behaviour was investigated in genetically selected Marchigian Sardinian alcohol preferring rats. These rats demonstrate an anxious phenotype and are highly sensitive to stress and stress-induced drinking. Key Results: Systemic administration of L822429 significantly reduced operant alcohol self-administration in Marchigian Sardinian alcohol preferring rats, but did not reduce alcohol self-administration in stock Wistar rats. NK1 receptor antagonism also attenuated yohimbine-induced reinstatement of alcohol seeking at all doses tested but had no effect on cue-induced reinstatement of alcohol seeking. L822429 reduced operant alcohol self-administration when injected into the lateral cerebroventricles or the medial amygdala. L822429 injected into the medial amygdala also significantly reduced anxiety-like behaviour in the elevated plus maze test. No effects on alcohol intake were observed following injection of L822429 into the dorsal or the ventral hippocampus. Conclusions and Implications Our results suggest that NK1 receptor antagonists may be useful for the treatment of alcohol addiction associated with stress or comorbid anxiety disorders. The medial amygdala appears to be an important brain site of action of NK1 receptor antagonism

Diversity of inhibitory and excitatory parvalbumin interneuron circuits in the dorsal horn

Parvalbumin-expressing interneurons (PVINs) in the spinal dorsal horn are found primarily in laminae II inner and III. Inhibitory PVINs (iPVINs) play an important role in segregating innocuous tactile input from pain-processing circuits through presynaptic inhibition of myelinated low-threshold mechanoreceptors and postsynaptic inhibition of distinct spinal circuits. By comparison, relatively little is known of the role of excitatory PVINs (ePVINs) in sensory processing. Here we use neuroanatomical and optogenetic approaches to show that ePVINs comprise a larger proportion of the PVIN population than previously reported, and that both ePVIN and iPVIN populations form synaptic connections amongst (and between) themselves. We find that these cells contribute to neuronal networks that influence activity within several functionally distinct circuits, and that aberrant activity of ePVINs under pathological conditions is well placed to contribute to the development of mechanical hypersensitivity