Cocaine self-administration in the mouse: A low-cost, chronic catheter preparation

Intravenous drug self-administration is the most valid animal model of human addiction because it allows volitional titration of the drug in the blood based on an individual’s motivational state together with the pharmacokinetic properties of the drug. Here we describe a reliable low-cost mouse self-administration catheter assembly and protocol that that can be used to assess a variety of drugs of abuse with a variety of protocols. We describe a method for intravenous catheter fabrication that allows for efficient and long-lasting intravenous drug delivery. The intravenous catheters remained intact and patent for several weeks allowing us to establish stable maintenance of cocaine acquisition. This was followed by a dose response study in the same mice. For collaborators interested in premade catheters for research please make a request at www.neuro-cloud.net/nature-precedings/pomerenze

High fidelity optogenetic control of individual prefrontal cortical pyramidal neurons in vivo

Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKll{\alpha}-ChR2-eYFP) or silence (AAV-CaMKll{\alpha}-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons in vivo. In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools combined with optrode recording methods provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior.Comment: 4 pages, 4 figures F1000Research articl

A High-Light Sensitivity Optical Neural Silencer: Development and Application to Optogenetic Control of Non-Human Primate Cortex

Technologies for silencing the electrical activity of genetically targeted neurons in the brain are important for assessing the contribution of specific cell types and pathways toward behaviors and pathologies. Recently we found that archaerhodopsin-3 from Halorubrum sodomense (Arch), a light-driven outward proton pump, when genetically expressed in neurons, enables them to be powerfully, transiently, and repeatedly silenced in response to pulses of light. Because of the impressive characteristics of Arch, we explored the optogenetic utility of opsins with high sequence homology to Arch, from archaea of the Halorubrum genus. We found that the archaerhodopsin from Halorubrum strain TP009, which we named ArchT, could mediate photocurrents of similar maximum amplitude to those of Arch (∼900 pA in vitro), but with a >3-fold improvement in light sensitivity over Arch, most notably in the optogenetic range of 1–10 mW/mm2, equating to >2× increase in brain tissue volume addressed by a typical single optical fiber. Upon expression in mouse or rhesus macaque cortical neurons, ArchT expressed well on neuronal membranes, including excellent trafficking for long distances down neuronal axons. The high light sensitivity prompted us to explore ArchT use in the cortex of the rhesus macaque. Optical perturbation of ArchT-expressing neurons in the brain of an awake rhesus macaque resulted in a rapid and complete (∼100%) silencing of most recorded cells, with suppressed cells achieving a median firing rate of 0 spikes/s upon illumination. A small population of neurons showed increased firing rates at long latencies following the onset of light stimulation, suggesting the existence of a mechanism of network-level neural activity balancing. The powerful net suppression of activity suggests that ArchT silencing technology might be of great use not only in the causal analysis of neural circuits, but may have therapeutic applications

Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation

Pain after disease/damage of the nervous system is predominantly treated with opioids, but without exploration of the long-term consequences. We demonstrate that a short course of morphine after nerve injury doubles the duration of neuropathic pain. Using genetic and pharmacological interventions, and innovative Designer Receptor Exclusively Activated by Designer Drugs disruption of microglia reactivity, we demonstrate that opioid-prolonged neuropathic pain arises from spinal microglia and NOD-like receptor protein 3 inflammasome formation/activation. Inhibiting these processes permanently resets amplified pain to basal levels, an effect not previously reported. These data support the “two-hit hypothesis” of amplification of microglial activation—nerve injury being the first “hit,” morphine the second. The implications of such potent microglial “priming” has fundamental clinical implications for pain and may extend to many chronic neurological disorders

Acute stress induces the rapid and transient induction of caspase-1, gasdermin D and release of constitutive IL-1 protein in dorsal hippocampus

The proinflammatory cytokine interleukin (IL)-1 plays a pivotal role in the behavioral manifestations (i.e., sickness) of the stress response. Indeed, exposure to acute and chronic stressors induces the expression of IL-1 in stress-sensitive brain regions. Thus, it is typically presumed that exposure to stressors induces the extracellular release of IL-1 in the brain parenchyma. However, this stress-evoked neuroimmune phenomenon has not been directly demonstrated nor has the cellular process of IL-1 release into the extracellular milieu been characterized in brain. This cellular process involves a form of inflammatory cell death, termed pyroptosis, which involves: 1) activation of caspase-1, 2) caspase-1 maturation of IL-1, 3) caspase-1 cleavage of gasdermin D (GSDMD), and 4) GSDMD-induced permeability of the cell membrane through which IL-1 is released into the extracellular space. Thus, the present study examined whether stress induces the extra-cellular release of IL-1 and engages the above cellular process in mediating IL-1 release in the brain. Male Sprague-Dawley rats were exposed to inescapable tailshock (IS). IL-1 extra-cellular release, caspase-1 activity and cleavage of GSDMD were measured in dorsal hippocampus. We found that exposure to IS induced a transient increase in the release of IL-1 into the extracellular space immediately after termination of the stressor. IS also induced a transient increase in caspase-1 activity prior to IL-1 release, while activation of GSDMD was observed immediately after termination of the stressor. IS also increased mRNA and protein expression of the ESCRTIII protein CHMP4B, which is involved in cellular repair. The present results suggest that exposure to an acute stressor induces the hallmarks of pyroptosis in brain, which might serve as a key cellular process involved in the release of IL-1 into the extracellular milieu of the brain parenchyma.</p

Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-μm), fixed tissue samples, using spinal cord sections. This method’s effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time

Optogenetic Mimicry of the Transient Activation of Dopamine Neurons by Natural Reward Is Sufficient for Operant Reinforcement

Activation of dopamine receptors in forebrain regions, for minutes or longer, is known to be sufficient for positive reinforcement of stimuli and actions. However, the firing rate of dopamine neurons is increased for only about 200 milliseconds following natural reward events that are better than expected, a response which has been described as a “reward prediction error” (RPE). Although RPE drives reinforcement learning (RL) in computational models, it has not been possible to directly test whether the transient dopamine signal actually drives RL. Here we have performed optical stimulation of genetically targeted ventral tegmental area (VTA) dopamine neurons expressing Channelrhodopsin-2 (ChR2) in mice. We mimicked the transient activation of dopamine neurons that occurs in response to natural reward by applying a light pulse of 200 ms in VTA. When a single light pulse followed each self-initiated nose poke, it was sufficient in itself to cause operant reinforcement. Furthermore, when optical stimulation was delivered in separate sessions according to a predetermined pattern, it increased locomotion and contralateral rotations, behaviors that are known to result from activation of dopamine neurons. All three of the optically induced operant and locomotor behaviors were tightly correlated with the number of VTA dopamine neurons that expressed ChR2, providing additional evidence that the behavioral responses were caused by activation of dopamine neurons. These results provide strong evidence that the transient activation of dopamine neurons provides a functional reward signal that drives learning, in support of RL theories of dopamine function

Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience. &nbsp;</p