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The medial prefrontal cortex (mPFC) is critical for learning and decision-making processes, including responding to threats. The protracted maturation of the mPFC extends into early adulthood. Although prominent models suggest that increasing top-down control by the mPFC eventually allows adult behavioral repertoires to emerge, it is unclear how progressive strengthening can produce nonlinear behavioral changes observed across development. We use fiber photometry and optogenetics to establish causal links between frontolimbic pathway activity and threat avoidance strategies in juvenile, adolescent and adult mice. We uncover multiple developmental switches in the roles of mPFC pathways targeting the nucleus accumbens and basolateral amygdala. These changes are accompanied by axonal pruning, strengthening of synaptic connectivity and altered functional connectivity with downstream cell types, which occur in the mPFC-basolateral amygdala and mPFC-nucleus accumbens pathways at different rates. Our results reveal how developing mPFC pathways pass through distinct architectures that may make them optimally adapted to age-specific challenges., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Motor output results from the coordinated activity of neural circuits distributed across multiple brain regions that convey information to the spinal cord via descending motor pathways. Yet the organizational logic through which supraspinal systems target discrete components of spinal motor circuits remains unclear. Here, using viral transsynaptic tracing along with serial two-photon tomography, we have generated a whole-brain map of monosynaptic inputs to spinal V1 interneurons, a major inhibitory population involved in motor control. We identified 26 distinct brain structures that directly innervate V1 interneurons, spanning medullary and pontine regions in the hindbrain as well as cortical, midbrain, cerebellar, and neuromodulatory systems. Moreover, we identified broad but biased input from supraspinal systems onto V1 Foxp2 and V1 Pou6f2 neuronal subsets. Collectively, these studies reveal elements of biased connectivity and convergence in descending inputs to molecularly distinct interneuron subsets and provide an anatomical foundation for understanding how supraspinal systems influence spinal motor circuits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Anxiety is an emotional state precipitated by the anticipation of real or potential threats. Anxiety disorders are the most prevalent psychiatric illnesses globally and increase the risk of developing comorbid conditions that negatively impact the brain and body. The etiology of anxiety disorders remains unresolved, limiting improvement of therapeutic strategies to alleviate anxiety-related symptoms with increased specificity and efficacy. Here, we applied novel intersectional tools to identify a discrete population of brainstem adrenergic neurons, named C1 cells, that promote aversion and anxiety-related behaviors via projections to the periaqueductal gray matter (PAG). While C1 cells have traditionally been implicated in modulation of autonomic processes, rabies tracing revealed that they receive input from brain areas with diverse functions. Calcium-based in vivo imaging showed that activation of C1 cells enhances excitatory responses in vlPAG, activity that is exacerbated in times of heightened stress. Furthermore, inhibition of C1 cells impedes the development of anxiety-like behaviors in response to stressful situations. Overall, these findings suggest that C1 neurons are positioned to integrate complex information from the brain and periphery for the promotion of anxiety-like behaviors.

As discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple features. Here we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), an adeno-associated virus-based, single-construct, intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes. ConVERGD offers benefits over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based features, accommodation of larger and more complex payloads and a vector that is easy to modify for rapid toolkit expansion. In the present report we employed ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE + locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Granulomatous hepatitis (GH) is a form of chronic hepatitis (CH) in dogs for which limited information is published., Hypothesis: Describe the clinical presentation, clinical pathology, ultrasound, and hepatic histopathology findings and to report survival times in dogs with GH., Animals: Twenty-nine client-owned dogs with GH., Methods: Retrospective observational study. Pathology records were searched. Inclusion criteria included a histopathologic diagnosis of GH, absence of an identified etiology or evidence of extrahepatic granulomatous disease, and a medical record available for review. Clinical presentation, clinical pathologic findings, treatment protocols, and survival times were recorded. Available hepatic biopsy material was graded and scored, and ultrasound evaluations reviewed., Results: The median age was 7 years (range, 0.66-12 years). Nineteen breeds were represented. Decreased appetite (19/29), lethargy (16/29), and fever (13/29) were seen most commonly. All dogs had increased serum transaminase activities, whereas 21/29 and 12/24 had hyperbilirubinemia and neutrophilia, respectively. Ultrasonographic findings included hepatomegaly (12/22), nodular parenchymal lesions (9/22), and hyperechoic parenchymal bands (8/22). Histopathologic necroinflammatory scores were moderate to severe in 16/19 dogs, and fibrosis scores were mild in 14/19 dogs. Treatments varied and included antibiotics, immunosuppressive drugs, and hepatoprotectants. Overall median survival was 635 days (range, 1-2482 days)., Conclusion and Clinical Importance: Granulomatous hepatitis in dogs is associated with high histopathologic grade, fever, neutrophilia, and a high incidence of hepatomegaly and focal parenchymal lesions on ultrasound examination. Despite disease severity on presentation, dogs with GH can have a good outcome with prolonged survival., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

In this issue of Cell Reports Methods, Jin et al. report the generation and validation of a rabies variant, RVΔL, for projection-based neuronal labeling. RVΔL shows little toxicity in vivo and has an improved growth advantage over another variant, RVΔGL, making it a useful tool for a wide variety of systems neuroscience-based studies., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The medial prefrontal cortex (mPFC) plays a key role in learning, mood and decision making, including in how individuals respond to threats 1-6 . mPFC undergoes a uniquely protracted development, with changes in synapse density, cortical thickness, long-range connectivity, and neuronal encoding properties continuing into early adulthood 7-21 . Models suggest that before adulthood, the slow-developing mPFC cannot adequately regulate activity in faster-developing subcortical centers 22,23 . They propose that during development, the enhanced influence of subcortical systems underlies distinctive behavioural strategies of juveniles and adolescents and that increasing mPFC control over subcortical structures eventually allows adult behaviours to emerge. Yet it has remained unclear how a progressive strengthening of top-down control can lead to nonlinear changes in behaviour as individuals mature 24,25 . To address this discrepancy, here we monitored and manipulated activity in the developing brain as animals responded to threats, establishing direct causal links between frontolimbic circuit activity and the behavioural strategies of juvenile, adolescent and adult mice. Rather than a linear strengthening of mPFC synaptic connectivity progressively regulating behaviour, we uncovered multiple developmental switches in the behavioural roles of mPFC circuits targeting the basolateral amygdala (BLA) and nucleus accumbens (NAc). We show these changes are accompanied by axonal pruning coinciding with functional strengthening of synaptic connectivity in the mPFC-BLA and mPFC-NAc pathways, which mature at different rates. Our results reveal how developing mPFC circuits pass through distinct architectures that may make them optimally adapted to the demands of age-specific challenges.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction., Competing Interests: Declaration of interests G.N. is a co-founder of Solgate., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aim: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function., Methods: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc)., Results: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8 -/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8 -/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues., Conclusion: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

As the discovery of cellular diversity in the brain accelerates, so does the need for functional tools that target cells based on multiple features, such as gene expression and projection target. By selectively driving recombinase expression in a feature-specific manner, one can utilize intersectional strategies to conditionally promote payload expression only where multiple features overlap. We developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches. ConVERGD offers benefits over existing platforms, such as expanded intersectionality, the ability to accommodate larger and more complex payloads, and a vector design that is easily modified to better facilitate rapid toolkit expansion. To demonstrate its utility for interrogating neural circuitry, we employed ConVERGD to target an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus (LC) identified via single-cell transcriptomic profiling to co-express the stress-related endogenous opioid gene prodynorphin ( Pdyn ). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn -expressing NE + LC neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Breathing, and the motor circuits that control it, is essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here, we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation in mice during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits., Competing Interests: AV, MM, ML, EB, RK, AA, LS, XD, NZ, LL, PP No competing interests declared, (© 2022, Vagnozzi et al.)

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.

Emotions are distinct patterns of behavioral and physiological responses triggered by stimuli that induce different brain states. Elucidating the circuits is difficult because of challenges in interrogating emotional brain states and their complex outputs. Here, we leverage the recent discovery in mice of a neural circuit for sighing, a simple, quantifiable output of various emotions. We show that mouse confinement triggers sighing, and this "claustrophobic" sighing, but not accompanying tachypnea, requires the same medullary neuromedin B (Nmb)-expressing neurons as physiological sighing. Retrograde tracing from the Nmb neurons identified 12 forebrain centers providing presynaptic input, including hypocretin (Hcrt)-expressing lateral hypothalamic neurons. Confinement activates Hcrt neurons, and optogenetic activation induces sighing and tachypnea whereas pharmacologic inhibition suppresses both responses. The effect on sighing is mediated by HCRT directly on Nmbneurons. We propose that this HCRT-NMB neuropeptide relay circuit mediates claustrophobic sighing and that activated Hcrt neurons are a claustrophobia brain state that directly controls claustrophobic outputs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The locus coeruleus (LC) is a seemingly singular and compact neuromodulatory nucleus that is a prominent component of disparate theories of brain function due to its broad noradrenergic projections throughout the CNS. As a diffuse neuromodulatory system, noradrenaline affects learning and decision making, control of sleep and wakefulness, sensory salience including pain, and the physiology of correlated forebrain activity (ensembles and networks) and brain hemodynamic responses. However, our understanding of the LC is undergoing a dramatic shift due to the application of state-of-the-art methods that reveal a nucleus of many modules that provide targeted neuromodulation. Here, we review the evidence supporting a modular LC based on multiple levels of observation (developmental, genetic, molecular, anatomical, and neurophysiological). We suggest that the concept of the LC as a singular nucleus and, alongside it, the role of the LC in diverse theories of brain function must be reconsidered., (Copyright © 2019 the authors.)

Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9 / Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain., (Copyright © 2017, American Association for the Advancement of Science.)

Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1 + or SF1 + cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.

The release of the neurotransmitter norepinephrine throughout the mammalian brain is important for modulating attention, arousal, and cognition during many behaviors. Furthermore, disruption of norepinephrine-mediated signaling is strongly associated with several psychiatric and neurodegenerative disorders in humans, emphasizing the clinical importance of this system. Most of the norepinephrine released in the brain is supplied by a very small, bilateral nucleus in the brainstem called the locus coeruleus. The goal of this minireview is to emphasize the complexity of the locus coeruleus beyond its primary definition as a norepinephrine-producing nucleus. Several recent studies utilizing innovative technologies highlight how the locus coeruleus-norepinephrine system can now be targeted with increased accuracy and resolution, in order to better understand its role in modulating diverse behaviors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity., (Copyright © 2014 the authors 0270-6474/14/3416637-13$15.00/0.)

Objective: To determine the prevalence of various radiographic findings for dogs with cardiac tamponade (CT) attributable to pericardial effusion (PE) and to determine the sensitivity and specificity of such findings for identification of affected dogs., Design: Retrospective, randomized, blinded, controlled study., Animals: 50 dogs with CT attributable to PE and 23 control dogs (10 healthy dogs and 13 dogs with cardiac diseases other than CT)., Procedures: Thoracic radiographic images of dogs were evaluated by an observer who was unaware of the dogs' medical histories. For each dog, a vertebral heart score, globoid appearance of the cardiac silhouette, and convexity of the dorsocaudal aspect of the cardiac silhouette were determined., Results: The sensitivity and specificity of enlargement of the cardiac silhouette (vertebral heart score, ≥ 10.7) for identification of dogs with CT attributable to PE were 77.6% and 47.8%, respectively. The sensitivity and specificity of a globoid appearance of the cardiac silhouette for identification dogs with CT were 41.9% and 40.0%, respectively. The sensitivity and specificity of a convex appearance of the dorsocaudal aspect of the cardiac silhouette for identification of dogs with CT were 57.1% and 35.0%, respectively., Conclusions and Clinical Relevance: Results of this study indicated none of the evaluated radiographic variables was highly (> 90%) sensitive or specific for identification of dogs with CT attributable to PE. Thoracic radiographic findings should not be considered reliable for identification of dogs with CT attributable to PE.

Extracellular signaling between cells is often transduced via receptors that reside at the cell membrane. In neurons this receptor-mediated signaling can promote a variety of cellular events such as differentiation, axon outgrowth and guidance, and synaptic development and function. Endocytic membrane trafficking of receptors ensures that the strength and duration of an extracellular signal is properly regulated. The covalent modification of membrane proteins by ubiquitin is a key biological mechanism controlling receptor internalization and endocytic sorting to recycling and degradative pathways in many cell types. In this review we highlight recent findings regarding the ubiquitin-dependent trafficking and turnover of receptors in neurons and the implications for neuronal development and function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Interaction with computer-based medical devices in the operating room is often challenging for surgeons due to sterility requirements and the complexity of interventional procedures. Typical solutions, such as delegating the interaction task to an assistant, can be inefficient. We propose a method for gesture-based interaction in the operating room that surgeons can customize to personal requirements and interventional workflow. Given training examples for each desired gesture, our system learns low-dimensional manifold models that enable recognizing gestures and tracking particular poses for fine-grained control. By capturing the surgeon's movements with a few wireless body-worn inertial sensors, we avoid issues of camera-based systems, such as sensitivity to illumination and occlusions. Using a component-based framework implementation, our method can easily be connected to different medical devices. Our experiments show that the approach is able to robustly recognize learned gestures and to distinguish these from other movements.

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Surgical stapling equipment was used to create a gastropexy in 20 dogs undergoing emergency surgery for gastric dilatation and volvulus (GDV). The technique involved creation of a tunnel between the seromuscular layer and the submucosa of the pyloric antrum, and a matching tunnel beneath the right m. transversus abdominis. The arms of a gastrointestinal anastomosis stapling device were introduced into the tunnels, and the device was fired to create the gastropexy. One dog died of systemic sequelae of GDV during the early postoperative period. None of the remaining 19 dogs developed a recurrence of GDV during follow-up periods ranging from 5 to 43 months. In 11 dogs, the integrity of the gastropexy was evaluated by abdominal ultrasonography and either negative contrast gastrography or double contrast gastrography; in these dogs, the radiographic and/or the ultrasonographic findings were suggestive of an intact gastropexy. There were no complications involving the gastropexy staple line. The results of this study indicate that an effective and consistent permanent gastropexy can be created, using surgical stapling equipment.

Background: In addition to the regulation of blood pressure, alpha2- and beta-adrenoceptor (AR) subtypes play an important role in the modulation of noradrenergic neurotransmission in the human CNS and PNS. Several studies suggest that the alpha2-AR responsiveness in cells and tissues after chronic epinephrine (EPI) or norepinephrine (NE) exposure may vary, depending on the beta-AR activity present there. Recently, we reported that in BE(2)-C human neuroblastoma cells (endogenously expressing alpha2A- and beta2-AR), chronic EPI treatment (300 nM) produced a dramatic beta-adrenoceptor-dependent desensitization of the alpha2A-AR response. The aim of this study is to determine if stable addition of a beta2-AR to a second neuroblastoma cell line (SH-SY5Y), that normally expresses only alpha2A-ARs that are not sensitive to 300 nM EPI exposure, would suddenly render alpha2A-ARs in that cell line sensitive to treatment with the same EPI concentration., Methods: These studies employed RT-PCR, receptor binding and inhibition of cAMP accumulation to confirm alpha2-AR subtype expression. Stable clones of SH-SY5Y cells transfected to stably express functional beta2-ARs (SHbeta2AR4) were selected to compare sensitivity of alpha2-AR to EPI in the presence or absence of beta2-ARs., Results: A series of molecular, biochemical and pharmacological studies indicated that the difference between the cell lines could not be attributed to alpha2-AR heterogeneity. We now report that after transfection of functional beta2-AR into SH-SY5Y cells (SHbeta2AR4), chronic treatment with modest levels of EPI desensitizes the alpha2A-AR. This effect results from a beta2-AR dependent down-regulation of native alpha2A-ARs by EPI accompanied by enhanced translocation of GRK2 and GRK3 to the membrane (required for GRK-mediated phosphorylation of agonist-occupied receptors)., Conclusion: This study further supports the hypothesis that the presence of the beta-AR renders the alpha2A-AR more susceptible to desensitization with physiological levels of EPI.

The anaerobic threshold (AT) has been defined as the theoretical highest exercise level that can be maintained for prolonged periods. It is of practical importance to the competitive endurance athlete to measure progress and plan training programs. The primary objective of this study was to assess the reliability and validity of breakpoint in the respiratory rate (RR) during incremental exercise as a marker for the AT. Secondary objectives were 1) to assess the reliability of the ventilatory threshold (VE) and ventilatory equivalent (VE/VO2) breakpoint, and 2) to assess differences in these 3 methods for their potential to measure change in fitness, as measured by standard error of measurement (SEM), coefficient of variability (CV), and correlation coefficient (R). Fifteen competitive male cyclists (5 category II, 6 category III, 1 category IV, 3 category V United States Cycling Federation) completed 2 maximal oxygen consumption tests within one week on an electronically braked cycle ergometer. A repeated measures Analysis of Variance using 2x3 design (test and methods) resulted in no significant differences (F = 0.02, p = 0.978), indicating that 1)all 3 methods are reproducible, and 2) RR, when compared to VE and VE/VO2, is a valid method of assessing the anaerobic threshold. The lowest SEM, lowest CV and highest R were obtained with the VE method (SEM = 19.4 watts, CV = 6.7%, R = 0.872), compared to VE/VO2 (SEM = 21.5 watts, CV = 7.4%, R=.811) and RR (SEM = 35.3 watts, CV = 12.2%, R = 0.800). From the results of this study, it is concluded that the RR method is a valid and reliable method for detecting AT. However, due to the relatively high SEM and CV, and low R, when compared to VE and VE/VO2, its insensitivity to small changes seen in highly fit athletes would preclude its use in measuring changes in AT. It appears that either VE or VE/VO2 would be appropriate for measuring AT changes in highly fit athletes. Key PointsRespiratory rate is a valid and reliable marker of the anaerobic threshold.Due to a relatively high standard error of measurement and coefficient of variability for the respiratory rate method, use of ventilation (VE) and ventilatory equivalent for oxygen (VE/VO2 is preferred when assessing changes in anaerobic threshold.When assessing changes in maximal aerobic capacity, maximal watts has a lower standard error of measurement and coefficient of variability and is preferred over changes in maximal oxygen consumption.

Chronic psychosocial stress markedly reduces the expression of high-frequency stimulation (HFS)-evoked early long-term potentiation (LTP) in the CA1 region of the hippocampus of anesthetized rats. Immunoblotting was performed to determine changes in molecular levels of key signaling proteins that might be responsible for this inhibitory effect. Western blot analysis of the CA1 region demonstrates that chronic psychosocial stress decreases basal levels of calcium calmodulin kinase II (CaMKII), phosphorylated (P)-CaMKII, calmodulin, and protein kinase C (PKCgamma) while markedly increasing protein phosphatase 2B (calcineurin) levels. The decrease of basal levels of P-CaMKII may be triggered primarily by excessive dephosphorylation resulting from enhanced basal levels of calcineurin. The decline in the basal levels of the upstream molecules, PKCgamma and calmodulin may be a consequence of the diminished basal P-CaMKII levels. Analysis of signaling molecules in CA1 region of chronically stressed rat subjected to HFS in vivo showed only one difference compared to similarly stimulated control rats; no increase in P-CaMKII levels. Our results suggest that decreased P-CaMKII levels may be primarily responsible for the stress-induced reduction in LTP expression.

Upper gastrointestinal examinations were performed in 11 unsedated ferrets and 4 ferrets sedated with ketamine and diazepam. Each animal received a 8-13 mL/kg body weight dosage of barium liquid (30% weight:volume). Radiographs were made immediately and at 5, 10, 20, 40, 60, 90, 120 and 150 min (mins) after the barium was administered. Gastric emptying began immediately. Mean total gastric emptying was longer in sedated ferrets (130 +/- 40 min versus 75 +/- 54 min); however, this difference was not statistically significant (p = 0.18). Small intestinal transit time was less than 2 h in all ferrets. The barium-filled small bowel was best visualized on the 20- and 40-min radiographs and did not exceed 5-7 mm in width. Flocculation of barium in the small intestine and adherence of barium to the stomach mucosa was seen in almost all animals. The longitudinal colonic mucosal folds in the colon were well visualized in the normal upper gastrointestinal study and aided in distinguishing small intestine from large intestine. The use of ketamine and diazepam sedation did not significantly affect the parameters evaluated in the upper gastrointestinal study series.

Chronic psychosocial stress impairs early long-term potentiation (LTP) in the hippocampal CA1 region but not in the dentate gyrus of anesthetized rats. Analysis of putative signaling molecules involved in the expression of LTP was performed to determine the possible reason(s) for the apparent resistance of the LTP of the dentate gyrus to chronic psychosocial stress. Immunoblotting was used to determine possible changes in the basal levels of various fractions of calcium-dependent calmodulin kinase II (CaMKII), phosphorylated CaMKII (P-CaMKII), calmodulin, protein kinase C gamma (PKCgamma) and calcineurin in the dentate gyrus of chronically stressed rats. Western blot analysis revealed that chronic stress significantly decreased the levels of the total CaMKII without affecting P-CaMKII levels. No significant change was detected in the levels of the upstream effectors, calmodulin and PKCgamma. However, chronic stress produced a significant decrease in calcineurin levels. The data suggest that the dentate gyrus of chronically stressed rats may have developed a compensatory mechanism whereby calcineurin levels are reduced to maintain normal P-CaMKII levels, which may be responsible for the normal early LTP of the dentate gyrus of chronically stressed rats. The results of this work will increase understanding of why certain brain regions are more resistant to deleterious effects of conditions that deteriorate learning and memory.

The anti-mitotic drugs colchicine and paclitaxel increase transfection efficiency of cationic liposomes. Using combined lipid-mediated transfection with anti-mitotic agents for gene therapy of cancer has been limited due to the likely development of multi-drug resistance (MDR). We treated human cancer cell lines and normal liver cells with glucocorticoids in combination with the antimitotics paclitaxel or colchicine before transient, cationic lipid-mediated transfection. Colchicine and paclitaxel each enhanced transgene expression in several cell lines. Moreover, glucocorticoid, combined with paclitaxel or colchicine, significantly increased reporter gene expression above that seen in cells treated with each drug alone. P-glycoprotein (PGP), a drug exporter encoded by ABCB1, exports both paclitaxel and colchicine. To determine the influence of PGP in colchicine- or paclitaxel-mediated enhancement of transgene expression, cells were treated with a histone deacetylase inhibitor, trichostatin A (TSA), known to induce ABCB1 expression, before treatment with colchicine or paclitaxel. TSA significantly reduced colchicine-mediated increases in reporter gene expression. Addition of glucocorticoid to colchicine pretreatment significantly attenuated TSA-mediated inhibition of colchicine-induced increases in transgene expression. TSA accelerated and glucocorticoid blocked export of rhodamine 123, a molecule known to be exported by PGP. The glucocorticoid/paclitaxel combination also increased reporter gene expression in BE(2)C cells, which constitutively express high levels of PGP. Thus, the degree of enhancement of transgene expression mediated by these anti-mitotics seems to be dependent on PGP activity. Glucocorticoids augment colchicine- or paclitaxel-mediated enhancement of transgene expression most likely by reducing drug egress through PGP.

This article illustrates the roles of ultrasonography, scintigraphy, and computed tomography (CT) as alternative techniques for pulmonary imaging in small animals. The advantages and limitations of each modality, normal anatomic features, and technical considerations will be discussed. Selected applications will be examined and include pulmonary consolidation, neoplasia and other masses, atelectasis, pneumothorax, dystrophic mineralization, diffuse infiltrative disease, and pulmonary embolism. The use of ultrasound and CT-guided interventional procedures will also be briefly discussed.

Historical, physical examination, clinicopathologic, radiographic and ultrasonographic findings of 13 dogs with hepatic abscesses were reviewed. Liver abscessation was characterized by number, size, shape, echogenicity and location. Solitary lesions greater than 3 cm were more common than multiple ones. The abscesses were mainly poorly echogenic lesions, often with central cavitation. The shape of the lesion ranged from round to oval or irregular. Enhancement artifact, abdominal effusion, regional lymphadenopathy and hyperechoic perihepatic fat, were identified in several dogs. Ultrasound-guided aspiration was performed in 10 of 13 dogs, and confirmed abscessation with cytologic and microbiologic evaluation. Ultrasound-guided percutaneous drainage of abscesses was performed as an adjunct to medical management in four dogs.

Aerosol delivery of gene therapy for treatment of lung diseases allows topical treatment of the airways with DNA concentrations not obtainable by systemic administration. We have investigated delivery of cationic liposomes complexed to plasmid DNA in a small particle aerosol. Plasmid cDNA-DMRIE/DOPE complexes were nebulized using either an Aerotech II or Puritan-Bennett 1600 (PB1600) nebulizer. Reservoir sampling showed that DNA-DMRIE/DOPE complexes were damaged to a significant degree during nebulization, such that activity of transfected gene was diminished. Of the nebulizers analyzed, DNA-DMRIE/DOPE complexes were more stable in the PB1600. The loss of effective transfection by DNA-DMRIE/DOPE, as detected by decreased reporter gene activity in A549 lung cells, was consistent with denaturation of the DMRIE/DOPE. In contrast, nebulized DNA-DOSPA/DOPE complexes retained complete ability to transfect. Adjustments to flow rate and reservoir volume of the PB1600 allowed a longer period of delivery of active DNA-DMRIE/DOPE particles. DNA-DMRIE/DOPE was radiolabeled with Technetium-99m (99mTc), nebulized, and the output captured in either an Andersen Sampler (AS) (Andersen, 1958) cascade impactor particle size analyzer or an all glass impinger. cDNA-cationic lipid complexes were detected in size ranges of 0.4-10 microns, with most particles found between 1-2 microns. Aerosol output was consistent from 0 to 5 min. These results show the feasibility of aerosol delivery of DNA-cationic lipids for the purposes of gene therapy to the lung.

Interferon regulatory factor-1 (IRF-1) gene expression is rapidly upregulated in the prolactin (PRL)-activated Nb2 rat T lymphoma cell line. To further elucidate its role as a T cell activation molecule, IRF-1 gene expression in response to various T cell stimuli was examined. In Nb2 T cells, PRL induced two peaks of IRF-1 gene expression: a rapid, transient peak at 1 h and a sustained peak at 12 h. PRL subsequently induced interferon-gamma (IFN-gamma) gene expression at 3-6 h. However, the early induction of IRF-1 and IFN-gamma does not appear to be interdependent. Interleukin-2 (IL-2) also induced IRF-1 gene expression in Nb2 T cells but only one broad peak at 10 h was observed. In primary mouse splenocytes, concanavalin A induced rapid and transient expression of the IRF-1 gene; maximal expression occurred by 6 h, and then returned to basal levels by 12-15 h. These results provide additional evidence for the importance of IRF-1 in T cell activation.

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.

The pituitary peptide hormone prolactin (Prl) is a potent inducer of Nb2 T lymphoma cell proliferation. To analyze the early genetic response to the mitogenic signals of Prl, a cDNA library was constructed from Nb2 T cells stimulated for 4 h with Prl and the protein synthesis inhibitor cycloheximide. Of 26 distinct clones isolated by differential screening, one clone, designated c25, exhibited extremely rapid but transient kinetics of induction by Prl and superinduction by Prl plus cycloheximide. Run-on transcription analysis indicated that c25 gene transcription was induced greater than 20-fold within 30 to 60 min of Prl stimulation. Surprisingly, DNA sequence analysis of c25 cDNA revealed that this Prl-inducible early-response gene is the rat homolog of the mouse transcription factor interferon-regulatory factor 1 (IRF-1), sharing 91% coding sequence similarity with mouse IRF-1. At the protein level, rat IRF-1 shares 97% and 92% homology with mouse IRF-1 and human IRF-1, respectively, suggesting that this molecule has been functionally conserved throughout evolution. Our studies show that the gene for IRF-1 is an immediate-early gene in Prl-stimulated T cells, which suggests that IRF-1 is a multifunctional molecule. In addition to its role in regulating growth-inhibitory interferon genes, IRF-1 may, therefore, also play a stimulatory role in cell proliferation. The gene for IRF-1 is one of the earliest genes known to be transcriptionally regulated by Prl.

Potentiation of interferon action was described as a non-additive, synergistic enhancement of interferon activity by combined preparations of immune and virus-type interferons. The potentiated antiviral activity was demonstrated for a variety of virus types as well as for the human and mouse in vitro systems. Potentiation by combined immune and fibroblast interferons was also demonstrated for the antitumor and direct anticellular effects of interferon. Use of partially purified immune and fibroblast interferons demonstrated that potentiation appeared to be a property of the interferon molecules themselves.

The neurotoxic agent, 3,3'-iminodiproprionitrile (IDPN), is a disrupter of neurofilament- and intermediate filament-organelle association. In the present study, the effect of IDPN on corticosteroidogenesis was investigated using isolated rat (having few intermediate filaments) and domestic fowl (having abundant intermediate filaments) adrenocortical cells. Cells were incubated with or without steroidogenic agents and precursors and with or without various concentrations of IDPN for 2 hr. IDPN had similar inhibitory potencies (as indicated by the half-maximal inhibitor concentrations (ID50 values] with both rat and domestic fowl cells despite their grossly different intermediate filament content. However, the average ID50 values of IDPN varied with the different steroidogenic agents and precursors used. The average IDPN ID50 values for maximal ACTH- and 8-bromo-cyclic AMP (8-Br-cAMP)-induced corticosterone production were equivalent (49.7 and 45.7 mM, respectively). However, the IDPN ID50 values for maximal ACTH-induced cAMP production, maximal 25-hydroxycholesterol- and pregnenolone-supported corticosterone production, and maximal ACTH- and 8-Br-cAMP-induced protein synthesis varied from 3.7 to 5.4 times the average ID50 values for maximal ACTH- and 8-Br-cAMP-induced corticosterone production. Thus, the inhibitory action of IDPN was not closely linked to the inhibition of ACTH-transmembrane signaling via cAMP, protein synthesis, and steroidogenic enzyme activity. The data suggest that IDPN inhibited corticosteroidogenesis at at a step after cAMP but before cholesterol side-chain cleavage and that the inhibition was not dependent on the presence of intermediate filaments.

Combination of interferon-gamma (IFN-gamma) with a mixture of IFN-alpha and IFN-beta (IFN-alpha/beta) produce a potentiated antiviral state. The onset of induction of the potentiated antiviral state was observed to be more rapid than the onset of induction of either IFN-gamma or IFN-alpha/beta separately. The onset of viral resistance to combined IFN-gamma and IFN-alpha/beta (25 U/ml each) began after 2 h, compared with 4 h for IFN-alpha/beta (25 U/ml) alone and 8 h for IFN-gamma (25 U/ml) alone. By adding actinomycin D at various times after interferon treatment, this more rapid onset of induction of the potentiated antiviral state was shown to be the result of a more rapid onset of cellular transcription. To determine whether increased concentrations of IFN-alpha/beta acted through a similar mechanism, we performed kinetic experiments with several concentrations of IFN-alpha/beta and with combined preparations of IFN-gamma and IFN-alpha/beta. The findings showed that treatment of cells with the combined interferons and with a high concentration of IFN-alpha/beta resulted in more rapid onset of cellular transcription and more rapid development of the antiviral state. Thus, the cellular response to potentiation appeared to involve perception by the cell of the combined interferons as a higher-than-expected effective concentration of interferon, resulting in more rapid onset of cellular transcription of mRNA for antiviral proteins.

The interaction of the interferons (IFNs) that cooperate to potentiate the antiviral action of IFN was studied. Serial dilutions of MuIFN-gamma and MuIFN-alpha/beta were employed separately and in combination to block virus replication in one-step, virus yield reduction experiments. To calculate the potentiation of IFN activity, protection levels obtained for each combination of MuIFN-gamma and MuIFN-alpha/beta were compared with those obtained for the separate IFNs. Potentiation levels increased with increasing concentrations of each of the IFNs in a dose-dependent manner, suggesting that potentiation of IFN's antiviral activity was the result of the mutually synergistic interaction of the IFNs. Three challenge viruses were employed: Mengo virus (positive-strand RNA virus), vesicular stomatitis virus (negative-strand RNA virus), and vaccinia virus (DNA virus). Identical results were observed with the three different viruses, suggesting that mutual synergism was a basic feature of the potentiation of IFN's antiviral activity by combined preparations of MuIFN-gamma and MuIFN-alpha/beta.

Potentiation was originally demonstrated as a nonadditive, synergistic enhancement of interferon (IFN) activity in the mouse system for mixed preparations containing MuIFN-alpha/beta and MuIFN-gamma. Potentiation of the antiviral and anticellular activities has now been studied for mouse and human systems, and in both systems IFN-alpha and IFN-beta interacted synergistically with IFN-gamma, but not with each other. Further, the antiviral and anticellular activities of IFN-alpha and IFN-beta were potentiated equally by IFN-gamma. Potentiation was demonstrated for HuIFNs with specific activities of 10(7) U/mg of protein and higher. Naturally produced and recombinant HuIFN-alpha s had the same relative abilities to be potentiated by HuIFN-gamma. It was concluded that IFN-gamma with either IFN-alpha or IFN-beta was essential for potentiation, that potentiation of anticellular and antiviral actions occurred in similar manners, and that a close correlation existed between potentiation in mouse and human systems. These results suggest that potentiation was caused by the interaction of two dissimilar IFN types (immune versus virus-type) and that potentiation studies in the mouse may be directly relevant for humans.