Subcellular specificity of cannabinoid effects in striatonigral circuits

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior

Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia

Metaplasticity of Hypothalamic Synapses following In Vivo Challenge

SummaryNeural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic α2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons

Fast glutamate and GABA transmission in Crh-IRES-Cre;Ai14 tdTomato neurons.

<p><b>A</b>) Left: Sample recording, in voltage-clamp mode, from a single tdTomato neuron in the presence of picrotoxin (100 µM) blocking GABAA receptors. Right: plots, obtained from analysis of 5 min recording showing a cumulative distribution of amplitudes (top) and inter event intervals (iei's) of spontaneous EPSCs during this period. <b>B</b>) Above: Averaged (black) and un-averaged (grey) evoked EPSCs. Paired-pulse interval is 50 msec. Below: Data from n = 12 tdTomato neurons showing paired-pulse ratio (PPR: evoke 2/evoke1). <b>C</b>) Left: Sample eEPSC traces recorded from an individual td+ cell at −80 mV (lower inward AMPAR-mediated current) and at +40 mV after addition of DNQX (10 µM; upper outward NMDAR current). Right: Ratio between inward AMPAR and outward NMDAR currents for n = 11 tdTomato neurons in the PVN. <b>D</b>) Left: spontaneous GABAAR-mediated inhibitory post-synaptic currents (IPSCs) recorded at −80 mV in a single td+ neuron with 10 µM DNQX. Right: cumulative distribution plots from this cell, of IPSC amplitudes and iei's. <b>E</b>) Above: evoked IPSCs with 50 msec interval. Averaged (black) and individual trials (grey) overlaid. Bottom: PPR data from n = 17 tdTomato neurons. <b>F</b>) Left: individual traces of evoked IPSCs (eIPSCs), in a td+ cell, recorded at varied holding potentials (−100 mV to −30 mV, 10 mV increment). Right: eIPSC current-voltage relationship showing eIPSC reversal potential in n = 6 td+ neurons. Data are represented by mean ± SEM. Scale bars 20 pA/50 msec in (<b>A,D</b>) and 50 pA/10 msec in (<b>B,C,E,F</b>).</p

Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus

The hypothalamus is a critical controller of homeostatic responses and plays a fundamental role in reward-seeking behaviour. Recently, hypothalamic neurones in the perifornical/lateral hypothalamic area (PF/LHA) have also been implicated in drug-seeking behaviour through projections to extra-hypothalamic sites such as the ventral tegmental area. For example, a population of neurones that expresses the peptide orexin has been strongly implicated in addiction-relevant behaviours. To date, the effect of addictive drugs on synaptic properties in the hypothalamus remains largely unexplored. Previous studies focusing on the PF/LHA neurones, however, have shown that the orexin system exhibits significant plasticity in response to food or sleep restriction. This neuroadaptive ability suggests that PF/LHA neurones could be highly susceptible to modifications by drug exposure. Here, we sought to determine whether cocaine produces synaptic plasticity in PF/LHA neurones. Whole-cell patch-clamp techniques were used to examine the effects of experimenter-administered (passive) or self-administered (SA) cocaine on glutamatergic synaptic transmission in PF/LHA neurones. These experiments demonstrate that both passive and SA cocaine exposure increases miniature excitatory postsynaptic current (mEPSC) frequency in PF/LHA neurones. In addition, SA cocaine reduced the paired-pulse ratio but the AMPA/NMDA ratio of evoked excitatory inputs was unchanged, indicative of a presynaptic locus for synaptic plasticity. Dual-labelling for orexin and excitatory inputs using the vesicular glutamate transporter (VGLUT2), showed that passive cocaine exposure increased VGLUT2-positive appositions onto orexin neurones. Further, a population of recorded neurones that were filled with neurobiotin and immunolabelled for orexin confirmed that increased excitatory drive occurs in this PF/LHA population. Given the importance of the PF/LHA and the orexin system in modulating drug addiction, we suggest that these cocaine-induced excitatory synapse-remodelling events within the hypothalamus may contribute to persistence in drug-seeking behaviour and relapse

Neurosecretory and neuropeptide phenotype of tdTomato cells in <i>Crh-IRES-Cre;Ai14</i> mice.

<p><b>A</b>) Retrograde transport of peripherally-delivered fluorogold in tdTomato neurons. A confocal image (40× magnification) shows tdTomato in the PVN (red) along with fluorogold immunoreactivity. Higher magnification (100×) image indicated by box is shown in lower left corner. Neuropeptide immunoreactivity for <b>B</b>) oxytocin (OT), <b>C</b>) vasopressin (VP), <b>D</b>) somatostatin (SST), or <b>E</b>) thyrotropin-releasing hormone (TRH; with 100× magnification inset) in <i>Crh-IRES-Cre;Ai14</i> mouse PVN, shown at 40× <b>F</b>) Bar graph showing the percent of tdTomato positive cells that coexpress each neuropeptide, each from n = 5 colchicine-treated mice. Graphed data are represented by mean ± SEM. Scale bars are 50 µm (A-E large), 20 µm (A, E inset).</p