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This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The ro...
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... The microarrays used for the nucleus accumbens n = 11 were constructed using 250 μm in the rows and 750 μm between the columns, with one column targeted at the nucleus accumbens core and the other targeting the nucleus accumbens shell. These arrays were lowered using a motorized stereotaxic instrument [8] at a rate 200 μm/min to a depth of 3.9 mm below the surface of the skull when implanting in the dorsal striatum, and a depth of 6.7 mm below the surface of the skull when implanting in the nucleus accumbens. Arrays were implanted through a rectangular craniotomy with the following corners (ML mm, AP mm) relative to bregma -DLS [(2.8, 2.5)(3.4, ...
The striatum, both dorsal and ventral, is strongly implicated in substance use disorder. Chronic consumption of abused substances, such as cocaine, can cause an oversaturation of mesostriatal dopamine, which results in alterations in the firing of striatal neurons. While most preclinical studies of drug self-administration (S-A) are focused on these alterations, individual differences in a subject's early responses to drugs can also account for substantial differences in addiction susceptibility. In this study, we modeled longitudinal pharmacokinetics using data from a previous longitudinal study (Coffey et al., 2015) and aimed to determine if firing in specific dorsal and ventral striatal subregions was subject to changes across chronic cocaine S-A, and if individual animal differences in striatal firing in response to early drug exposure correlated with increases in drug intake. We observed that the firing patterns of nucleus accumbens (NAc) core and shell neurons exhibited increasing sensitivity to cocaine over the first 6 S-A sessions and maintained a strong negative correlation between drug intake and neuronal firing rates across chronic S-A. Moreover, we observed that the early sensitivity of NAc shell neurons to cocaine correlated with future increases in drug intake. Specifically, rats whose NAc shell neurons were most inhibited by increasing levels of cocaine upon first exposure exhibited the strongest increases in cocaine intake over time. If this difference can be linked to a genetic difference, or druggable targets, it may be possible to screen for similar addiction susceptibility in humans or develop novel preemptive pharmacotherapies.
... For fiber photometry surgeries, rats were anesthetized with 1-3% isoflurane, and operated on using a custom robotic stereotaxic instrument (Coffey et al., 2013). Animals received 500nL injections of pGP-AAV1-syn-jGCaMP7f-WPRE (#104488, Addgene) at 100nL/min into the right Lateral Habenula (LHb; Males: A/P -3.7, M/L 0.95, D/V -5.2; Females: scaled*0.95) ...
Unlabelled:
Opioid use disorder has become an epidemic in the United States, fueled by the widespread availability of fentanyl, which produces rapid and intense euphoria followed by severe withdrawal and emotional distress. We developed a new preclinical model of fentanyl seeking in outbred male and female rats using volitional oral self-administration that can be readily applied in labs without intravascular access. Using a traditional two lever operant procedure, rats learned to take oral fentanyl vigorously, escalated intake across sessions, and readily reinstated responding to conditioned cues after extinction. Oral self-administration also revealed individual and sex differences that are essential to studying substance use risk propensity. During a behavioral economics task, rats displayed inelastic demand curves and maintained stable intake across a wide range of fentanyl concentrations. Oral SA was also neatly patterned, with distinct "loading" and "maintenance" phases of responding within each session. Using our software DeepSqueak, we analyzed thousands of ultrasonic vocalizations (USVs), which are innate expressions of current emotional state in rats. Rats produced 50 kHz USVs during loading then shifted quickly to 22 kHz calls despite ongoing maintenance oral fentanyl taking, reflecting a transition to negative reinforcement. Using fiber photometry, we found that the lateral habenula differentially processed drug-cues and drug-consumption depending on affective state, with potentiated modulation by drug cues and consumption during the negative affective maintenance phase. Together, these results indicate a rapid progression from positive to negative reinforcement occurs even within an active drug taking session, revealing a within-session opponent process.
Significance statement:
The United States opioid epidemic is defined by rampant and treatment resistant fentanyl use. Better understanding of neural substrates underlying this phenomenon is essential to slowing the opioid crisis. Intravenous and vapor self-administration (SA) are the standard models for studying fentanyl use in rodents, however they many carry pragmatic downsides. Here, we used a novel oral fentanyl self-administration model that provides key translational and technical benefits and can be readily applied in other labs to study the neurobiology of fentanyl SA. This method captured individual and sex differences necessary for studying substance use risk propensity and uncovered a rapid shift in affective state in rats, suggesting and shift from positive to negative reinforcement within each fentanyl taking session.
... For stereotaxic surgeries, anesthesia was induced with 3% isoflurane/97% oxygen and maintained at 1% isoflurane during the surgical procedure. Using a custom robotic stereotaxic instrument 44 , mice were injected with AAV8-DIO-hM 3 Dq-2a-RiboTag (n=32), AAV8-DIO-hM 3 Dq-mcherry (n=10), a 50/50 combination of AAV8-DIO-hM 3 Dq-mcherry and AAV8-hSyn-DIO-RiboTag (n=13), AAV8-hSyn-DIO-RiboTag alone (n=44), or AAV1-DIO-Synaptophysin-GFP (n=3) into LHb. Blunt 28g needles were inserted bilaterally, terminating at A/P −1.85, M/L ±0.35, and D/V −2.59, and 0.5 μl of the virus was injected at a rate of 0.2 μl/min. ...
The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase‐activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre‐dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal‐associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP‐expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM3Dq DREADD selectively in LHb PACAP‐expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole—they decreased anxiety‐like and fear behavior and produced a conditioned place preference. In conclusion, PACAP‐expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events. While LHb PACAP‐expressing neurons do not define a distinct phenotypic class of LHb neurons, they are unique in behavioral control. Activation of these neurons reduces fear and anxiety and is directly rewarding.
... All Salmon and DESeq2 settings were left default and our analysis pipeline is archived on our Galaxy server-http://172. 25 ...
... A new cohort of 15 animals (7 male, 8 female) were implanted with guide cannula terminating 0.5 mm above the dorsal raphe nucleus at a 15°angle (AP −4.5, ML 0.25, DV −2.75), using an automated stereotaxic instrument [25]. For cannulation surgeries, mice were anesthetized with 1-3% isoflurane. ...
Serotonin is a key mediator of stress, anxiety, and depression, and novel therapeutic targets within serotonin neurons are needed to combat these disorders. To determine how stress alters the translational profile of serotonin neurons, we sequenced ribosome-associated RNA from these neurons after repeated stress in male and female mice. We identified numerous sex- and stress-regulated genes. In particular, Fkbp5 mRNA, which codes for the glucocorticoid receptor co-chaperone protein FKBP51, was consistently upregulated in male and female mice following stress. Pretreatment with a selective FKBP51 inhibitor into the dorsal raphe prior to repeated forced swim stress decreased resulting stress-induced anhedonia. Our results support previous findings linking FKBP51 to stress-related disorders and provide the first evidence suggesting that FKBP51 function may be an important regulatory node integrating circulating stress hormones and serotonergic regulation of stress responses.
... Given the considerable manual dexterity required to perform procedures such as a craniectomy, bone thinning, as well as electrode or lens placement, a significant training period is required for technical mastery. Therefore, robotic assistants have been developed to aid in cranial remodeling (Coffey et al., 2013;Ghanbari et al., 2019b;Liang et al., 2019;Pak et al., 2015;Ramrath et al., 2009;Rynes et al., 2020). These tools should reduce experimental variability and facilitate adoption of cranial window studies. ...
Deciphering neurologic function is a daunting task, requiring understanding the neuronal networks and emergent properties that arise from the interactions among single neurons. Mechanistic insights into neuronal networks require tools that simultaneously assess both single neuron activity and the consequent mesoscale output. The development of cranial window technologies, in which the skull is thinned or replaced with a synthetic optical interface, has enabled monitoring neuronal activity from subcellular to mesoscale resolution in awake, behaving animals when coupled with advanced microscopy techniques. Here we review recent achievements in cranial window technologies, appraise the relative merits of each design and discuss the future research in cranial window design.
... For intersectional surgeries, anesthesia was induced with 5% isoflurane/95% oxygen and maintained at 1-3% isoflurane during the surgical procedure. Using a custom robotic stereotaxic instrument (Coffey et al., 2013), 27 animals received AAV8-hSyn-DIO-RiboTag injected into LHb. Blunt 28 g needles were inserted bilaterally at a 10 • angle terminating at A/P − 3.2, M/L ±0.7, and D/V − 5.25 and 1 μl of AAV8-hSyn-DIO-RiboTag was injected at a rate of 0.2 μl/min. ...
The lateral habenula (LHb) integrates critical information regarding aversive stimuli that shapes decision making and behavioral responses. The three major LHb outputs innervate dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). LHb neurons that project to these targets are segregated and nonoverlapping, and this led us to consider whether they have distinct molecular phenotypes and adaptations to stress exposure. In order to capture a time-locked profile of gene expression after repeated forced swim stress, we used intersectional expression of RiboTag in rat LHb neurons and next-gen RNA sequencing to interrogate the RNAs actively undergoing translation from each of these pathways. The “translatome” in the neurons comprising these pathways was similar at baseline, but diverged after stress, especially in the neurons projecting to the RMTg. Using weighted gene co-expression network analysis, we found one module, which had an overrepresentation of genes associated with phosphoinositide 3 kinase (PI3K) signaling, comprising genes downregulated after stress in the RMTg-projecting LHb neurons. Reduced PI3K signaling in RMTg-projecting LHb neurons may be a compensatory adaptation that alters the functional balance of LHb outputs to GABAergic vs. monoaminergic neurons following repeated stress exposure.
... All Salmon and DESeq2 settings were left default and our analysis pipeline is archived on our Galaxy server-http://172. 25 ...
... A new cohort of 15 animals (7 male, 8 female) were implanted with guide cannula terminating 0.5 mm above the dorsal raphe nucleus at a 15°angle (AP −4.5, ML 0.25, DV −2.75), using an automated stereotaxic instrument [25]. For cannulation surgeries, mice were anesthetized with 1-3% isoflurane. ...
... For intersectional surgeries, rats were anesthetized with 1-3% isoflurane. Using a custom robotic stereotaxic instrument [32], 58 animals received hM 4 Di injected into the LHb. Blunt 28 g needles were inserted bilaterally at a 10°angle terminating at A/P −3.2, M/L ± 0.7, and D/V −5.25 and 1 μl of AAV8-hSyn-DIO-hM 4 Di-mCherry was injected at a rate of 0.2 μl/min. ...
The lateral habenula (LHb) processes information about aversive experiences that contributes to the symptoms of stress disorders. Previously, we found that chemogenetic inhibition of rat LHb neurons reduced immobility in the forced swim test, but the downstream target of these neurons was not known. Using an intersectional viral vector strategy, we selectively transduced three different output pathways from the LHb by injecting AAV8-DIO-hM4Di into the LHb and CAV2-CRE (a retrograde viral vector) into one of the three target areas as follows: dorsal raphe nucleus (DRN), ventral tegmental area (VTA), or rostromedial tegmentum (RMTg). Using the forced swim test, we found that chemogenetic inhibition of DRN-projecting LHb neurons reduced passive coping (immobility), whereas inhibition of the other pathways did not. Chemogenetic activation of DRN-projecting neurons using hM3Dq in another cohort did not further exacerbate immobility. We next examined the impact of inhibiting DRN-projecting LHb neurons on reward sensitivity, perseverative behavior, and anxiety-like behavior using saccharin preference testing, reward-omission testing, and open-field testing, respectively. There was no effect of inhibiting any of these pathways on reward sensitivity, locomotion, or anxiety-like behavior, but inhibiting DRN-projecting LHb neurons reduced perseverative licking during reward-omission testing, whereas activating these neurons increased perseverative licking. These results support the idea that inhibiting LHb projections to the DRN provides animals with resilience during highly stressful or frustrating conditions but not under low-stress circumstances, and that inhibiting these neurons may promote persistence in active coping strategies.
... Consequently, this will lead to higher inter-subject variability. Although there are commercially available automated stereotaxic instruments (Pak et al., 2015) and open-source robotic instruments for craniotomy (Coffey et al., 2013;Ghanbari et al., 2018;Pak et al., 2015), these instruments cannot be readily adapted for tissue aspiration purposes. ...
... Open-source automated tools for biological experiments have become increasingly desirable to enhance experimental accuracy and efficiency (Ardesch et al., 2017;Bolanos et al., 2017;Coffey et al., 2013;Giovannucci et al., 2018;Pak et al., 2015). It can also reduce training time and unnecessary manpower. ...
... It can also reduce training time and unnecessary manpower. Recently, high precision automated surgical stereotaxic tools for craniotomies have been developed (Coffey et al., 2013;Ghanbari et al., 2018;Pak et al., 2015). These tools provided low-cost and easy-to-adopt solutions for a variety of surgeries such as cranial window/hole drilling, and skull thinning. ...
Background:
Gradient index (GRIN) lenses can be used to image deep brain regions otherwise inaccessible via standard optical imaging methods. Brain tissue aspiration before GRIN lens implantation is a widely adopted approach. However, typical brain tissue aspiration methods still rely on a handheld vacuum needle, which is subject to human error and low reproducibility. Therefore, a high-precision automated surgical instrument for brain tissue aspiration is desirable.
New method:
We developed a robotic surgical instrument that utilizes robotic control of a needle connected to a vacuum pump to aspirate brain tissue. The system was based on a commercial stereotaxic instrument, and the additional parts can be purchased off-the-shelf or Computer Numerical Control (CNC) machined. A MATLAB-based user-friendly graphical user interface (GUI) was developed to control the instrument.
Results:
We demonstrated the GRIN lens implantation procedure in the dorsal striatum utilizing our proposed surgical instrument and confirmed the surgical results by microscope after the implantation.
Compare with existing method(s):
Compared to the traditional handheld method, the automatic tissue aspiration can be performed by interacting with GUI. The instrument was designed specifically for microendoscope implantation, but it can also be easily adapted for robotic craniotomy. This robotic surgical instrument can minimize human error, reduce training time, and greatly increase surgical precision.
Conclusions:
Our robotic surgical instrument is an ideal solution for brain tissue aspiration prior to GRIN lens implantation. It will be useful for neuroscientists performing in vivo deep brain imaging using miniature microscope or two-photon microscope combined with microendoscopes.
... Male and female mice (25-30 g) were anesthetized using pentobarbital (50 mg/kg IP), and anesthesia was periodically maintained with a Ketamine/Xylazine cocktail (87.5 mg/kg Ketamine; 12.5 mg/kg Xylazine). Mice were surgically prepared for chronic, extracellular single unit recording as described previously (Barker et al., 2014) using a fully automated stereotaxic instrument (Coffey et al., 2013). Each animal was intracranially injected with 1 lL of AAV-EF1a-FLEX-Chronos-GFP (titer > 3 9 10 12 ) at the following coordinates relative to bregma over 10 min (ML 2.5 mm, AP 0.5 mm, DV À2.75 mm; right side). ...
... The microwires extended 300 lm ventral to the tip of the optical fiber and a maximum of 500 lm laterally from the center of the optical fiber, allowing for illumination of the entire wire array (Fig. 1a). Opto-arrays were lowered using a motorized stereotaxic device (Coffey et al., 2013) at a rate of 200 lm/min and to a depth of 2.75 mm below the surface of the skull. The non-driveable array was sealed to the surface of the skull with cyanoacrylate. ...
Striatal medium spiny projection neurons (MSNs) output through two diverging circuits, the “direct and indirect pathways” which originate from minimally overlapping populations of MSNs expressing either the dopamine receptor D1 or the dopamine receptor D2. One modern theory of direct and indirect pathway function proposes that activation of direct pathway MSNs facilitates output of desired motor programs, while activation of indirect pathway MSNs inhibits competing motor programs. A separate theory suggests that coordinated timing or synchrony of the direct and indirect pathways is critical for the execution of refined movements. These hypotheses are made testable by a common type of striatal neuron known as type IIb MSNs. Clusters of these MSNs exhibit phasic increases in firing rate related to sensorimotor activity of single body parts. If these MSNs were to reside in only the direct pathway, evidence would be provided that D1 MSNs are “motor program” specific, which would lend credence to the “competing motor programs” hypothesis. However, id type IIb reside in both pathways, evidence would be provided for the “coordinated timing or synchrony” hypothesis. Our results show that type IIb neurons may express either D1 or D2. This evidence supports the theory that the coordinated timing or synchrony of the direct and indirect pathways is critical for refined movements. We also propose a model in which the direct and indirect pathways act as a differentiator circuit, providing a possible mechanism by which coordinated activity of D1 and D2 neurons may output meaningful somatosensorimotor information to downstream structures.
