Rectal swab DNA collection protocol for PCR genotyping in rats.

DNA collection is essential for genotyping laboratory animals. Common collection methods require tissue amputation, causing discomfort and injury. Rectal swabbing has been proposed as an effective, minimally invasive alternative, but an evidence-backed protocol for the technique remains unavailable. This report evaluates the effects of collection parameters on the quality of PCR results and presents a protocol for genotyping a litter of rats within 3-5 h. Samples with 2-8 scrapes produced enough DNA to amplify targets up to ∼1800 bp long using PCR. Rectal swabbing produced PCR results with similar utility as ear clip samples, and results were unaffected by residual fecal matter or cell debris. The protocol enables fast, minimally invasive and repeatable genotyping using commercial PCR reagents.

DNA collection for genotyping laboratory rodents commonly requires tissue amputation, leading to injury and discomfort. Rectal swabbing can be an effective, minimally invasive alternative, but there is a lack of information on how to apply the technique and the necessary parameters involved. This report assesses collection and processing parameters while combining the rectal swab collection method with commercial PCR reagents to allow for fast genotyping with minimally processed DNA samples. Findings were used to design a protocol for minimally invasive DNA collection and genotyping.

Rectal Swab DNA Collection Protocol for PCR Genotyping in Rats.

DNA collection is essential for genotyping laboratory animals. However, common collection methods require tissue amputation, causing discomfort and injury. Rectal swabbing has been proposed as an effective non-invasive alternative, but an evidence-backed protocol for the technique remains unavailable. We evaluate the effect of collection parameters on PCR result quality and present a genotyping protocol that can yield results for a litter of rats within 3-5 hours. We found that samples with 2-8 scrapes produced enough DNA to amplify targets up to ~1800bp long using PCR. Rectal swabbing produced PCR results with similar quality to ear clip samples, and results were unaffected by residual fecal matter or cell debris. Our protocol enables fast, non-invasive, and repeatable genotyping using commercial PCR reagents.

Prefrontal cortical activity predicts the occurrence of nonlocal hippocampal representations during spatial navigation.

The receptive field of a neuron describes the regions of a stimulus space where the neuron is consistently active. Sparse spiking outside of the receptive field is often considered to be noise, rather than a reflection of information processing. Whether this characterization is accurate remains unclear. We therefore contrasted the sparse, temporally isolated spiking of hippocampal CA1 place cells to the consistent, temporally adjacent spiking seen within their spatial receptive fields ("place fields"). We found that isolated spikes, which occur during locomotion, are strongly phase coupled to hippocampal theta oscillations and transiently express coherent nonlocal spatial representations. Further, prefrontal cortical activity is coordinated with and can predict the occurrence of future isolated spiking events. Rather than local noise within the hippocampus, sparse, isolated place cell spiking reflects a coordinated cortical-hippocampal process consistent with the generation of nonlocal scenario representations during active navigation.

Optogenetic pacing of medial septum parvalbumin-positive cells disrupts temporal but not spatial firing in grid cells.

Grid cells in the medial entorhinal cortex (MEC) exhibit remarkable spatial activity patterns with spikes coordinated by theta oscillations driven by the medial septal area (MSA). Spikes from grid cells progress relative to the theta phase in a phenomenon called phase precession, which is suggested as essential to create the spatial periodicity of grid cells. Here, we show that optogenetic activation of parvalbumin-positive (PV+) cells in the MSA enabled selective pacing of local field potential (LFP) oscillations in MEC. During optogenetic stimulation, the grid cells were locked to the imposed pacing frequency but kept their spatial patterns. Phase precession was abolished, and speed information was no longer reflected in the LFP oscillations but was still carried by rate coding of individual MEC neurons. Together, these results support that theta oscillations are not critical to the spatial pattern of grid cells and do not carry a crucial velocity signal.

Transcutaneous Afferent Patterned Stimulation Therapy Reduces Hand Tremor for One Hour in Essential Tremor Patients.

Essential tremor (ET) patients often experience hand tremor that impairs daily activities. Non-invasive electrical stimulation of median and radial nerves in the wrist using a recently developed therapy called transcutaneous afferent patterned stimulation (TAPS) has been shown to provide symptomatic tremor relief in ET patients and improve patients' ability to perform functional tasks, but the duration of tremor reduction is unknown. In this single-arm, open-label study, fifteen ET patients performed four hand tremor-specific tasks (postural hold, spiral drawing, finger-to-nose reach, and pouring) from the Fahn-Tolosa-Marin Clinical Rating Scale (FTM-CRS) prior to, during, and 0, 30, and 60 min following TAPS. At each time point, tremor severity was visually rated according to the FTM-CRS and simultaneously measured by wrist-worn accelerometers. The duration of tremor reduction was assessed using (1) improvement in the mean FTM-CRS score across all four tasks relative to baseline, and (2) reduction in accelerometer-measured tremor power relative to baseline for each task. Patients were labeled as having at least 60 min of therapeutic benefit from TAPS with respect to each specified metric if all three (i.e., 0, 30, and 60 min) post-therapy measurements were better than that metric's baseline value. The mean FTM-CRS scores improved for at least 60 min beyond the end of TAPS for 80% (12 of 15, p = 4.6e-9) of patients. Similarly, for each assessed task, tremor power improved for at least 60 min beyond the end of TAPS for over 70% of patients. The postural hold task had the largest reduction in tremor power (median 5.9-fold peak reduction in tremor power) and had at least 60 min of improvement relative to baseline beyond the end of TAPS therapy for 73% (11 of 15, p = 9.8e-8) of patients. Clinical ratings of tremor severity were correlated to simultaneously recorded accelerometer-measured tremor power (r = 0.33-0.76 across the four tasks), suggesting tremor power is a valid, objective tremor assessment metric that can be used to track tremor symptoms outside the clinic. These results suggest TAPS can provide reductions in upper limb tremor symptoms for at least 1 h post-therapy in some patients, which may improve patients' ability to perform tasks of daily living.

Knock-In Rat Lines with Cre Recombinase at the Dopamine D1 and Adenosine 2a Receptor Loci.

Genetically modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here, we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways, respectively. We used whole-genome sequencing to confirm the lack of off-target effects and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

Specific hippocampal representations are linked to generalized cortical representations in memory.

Memories link information about specific experiences to more general knowledge that is abstracted from and contextualizes those experiences. Hippocampal-cortical activity patterns representing features of past experience are reinstated during awake memory reactivation events, but whether representations of both specific and general features of experience are simultaneously reinstated remains unknown. We examined hippocampal and prefrontal cortical firing patterns during memory reactivation in rats performing a well-learned foraging task with multiple spatial paths. We found that specific hippocampal place representations are preferentially reactivated with the subset of prefrontal cortical task representations that generalize across different paths. Our results suggest that hippocampal-cortical networks maintain links between stored representations for specific and general features of experience, which could support abstraction and task guidance in mammals.

Distinct hippocampal-cortical memory representations for experiences associated with movement versus immobility.

While ongoing experience proceeds continuously, memories of past experience are often recalled as episodes with defined beginnings and ends. The neural mechanisms that lead to the formation of discrete episodes from the stream of neural activity patterns representing ongoing experience are unknown. To investigate these mechanisms, we recorded neural activity in the rat hippocampus and prefrontal cortex, structures critical for memory processes. We show that during spatial navigation, hippocampal CA1 place cells maintain a continuous spatial representation across different states of motion (movement and immobility). In contrast, during sharp-wave ripples (SWRs), when representations of experience are transiently reactivated from memory, movement- and immobility-associated activity patterns are most often reactivated separately. Concurrently, distinct hippocampal reactivations of movement- or immobility-associated representations are accompanied by distinct modulation patterns in prefrontal cortex. These findings demonstrate a continuous representation of ongoing experience can be separated into independently reactivated memory representations.

Hippocampal-cortical interaction in decision making.

When making a decision it is often necessary to consider the available alternatives in order to choose the most appropriate option. This deliberative process, where the pros and cons of each option are considered, relies on memories of past actions and outcomes. The hippocampus and prefrontal cortex are required for memory encoding, memory retrieval and decision making, but it is unclear how these areas support deliberation. Here we examine the potential neural substrates of these processes in the rat. The rat is a powerful model to investigate the network mechanisms underlying deliberation in the mammalian brain given the anatomical and functional conservation of its hippocampus and prefrontal cortex to other mammalian systems. Importantly, it is amenable to large scale neural recording while performing laboratory tasks that exploit its natural decision-making behavior. Focusing on findings in the rat, we discuss how hippocampal-cortical interactions could provide a neural substrate for deliberative decision making.

Dopamine neurons modulate pheromone responses in Drosophila courtship learning.

Learning through trial-and-error interactions allows animals to adapt innate behavioural 'rules of thumb' to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.

Neuronal control of Drosophila courtship song.

The courtship song of the Drosophila male serves as a genetically tractable model for the investigation of the neural mechanisms of decision-making, action selection, and motor pattern generation. Singing has been causally linked to the activity of the set of neurons that express the sex-specific fru transcripts, but the specific neurons involved have not been identified. Here we identify five distinct classes of fru neuron that trigger or compose the song. Our data suggest that P1 and pIP10 neurons in the brain mediate the decision to sing, and to act upon this decision, while the thoracic neurons dPR1, vPR6, and vMS11 are components of a central pattern generator that times and shapes the song's pulses. These neurons are potentially connected in a functional circuit, with the descending pIP10 neuron linking the brain and thoracic song centers. Sexual dimorphisms in each of these neurons may explain why only males sing.

Sexual dimorphism in the fly brain.

BACKGROUND: Sex-specific behavior may originate from differences in brain structure or function. In Drosophila, the action of the male-specific isoform of fruitless in about 2000 neurons appears to be necessary and sufficient for many aspects of male courtship behavior. Initial work found limited evidence for anatomical dimorphism in these fru+ neurons. Subsequently, three discrete anatomical differences in central brain fru+ neurons have been reported, but the global organization of sex differences in wiring is unclear. RESULTS: A global search for structural differences in the Drosophila brain identified large volumetric differences between males and females, mostly in higher brain centers. In parallel, saturating clonal analysis of fru+ neurons using mosaic analysis with a repressible cell marker identified 62 neuroblast lineages that generate fru+ neurons in the brain. Coregistering images from male and female brains identified 19 new dimorphisms in males; these are highly concentrated in male-enlarged higher brain centers. Seven dimorphic lineages also had female-specific arbors. In addition, at least 5 of 51 fru+ lineages in the nerve cord are dimorphic. We use these data to predict >700 potential sites of dimorphic neural connectivity. These are particularly enriched in third-order olfactory neurons of the lateral horn, where we provide strong evidence for dimorphic anatomical connections by labeling partner neurons in different colors in the same brain. CONCLUSION: Our analysis reveals substantial differences in wiring and gross anatomy between male and female fly brains. Reciprocal connection differences in the lateral horn offer a plausible explanation for opposing responses to sex pheromones in male and female flies.

Cellular organization of the neural circuit that drives Drosophila courtship behavior.

BACKGROUND: Courtship behavior in Drosophila has been causally linked to the activity of the heterogeneous set of ∼1500 neurons that express the sex-specific transcripts of the fruitless (fru) gene, but we currently lack an appreciation of the cellular diversity within this population, the extent to which these cells are sexually dimorphic, and how they might be organized into functional circuits. RESULTS: We used genetic methods to define 100 distinct classes of fru neuron, which we compiled into a digital 3D atlas at cellular resolution. We determined the polarity of many of these neurons and computed their likely patterns of connectivity, thereby assembling them into a neural circuit that extends from sensory input to motor output. The cellular organization of this circuit reveals neuronal pathways in the brain that are likely to integrate multiple sensory cues from other flies and to issue descending control signals to motor circuits in the thoracic ganglia. We identified 11 anatomical dimorphisms within this circuit: neurons that are male specific, are more numerous in males than females, or have distinct arborization patterns in males and females. CONCLUSIONS: The cellular organization of the fru circuit suggests how multiple distinct sensory cues are integrated in the fly's brain to drive sex-specific courtship behavior. We propose that sensory processing and motor control are mediated through circuits that are largely similar in males and females. Sex-specific behavior may instead arise through dimorphic circuits in the brain and nerve cord that differentially couple sensory input to motor output.

BrainGazer--visual queries for neurobiology research.

Neurobiology investigates how anatomical and physiological relationships in the nervous system mediate behavior. Molecular genetic techniques, applied to species such as the common fruit fly Drosophila melanogaster, have proven to be an important tool in this research. Large databases of transgenic specimens are being built and need to be analyzed to establish models of neural information processing. In this paper we present an approach for the exploration and analysis of neural circuits based on such a database. We have designed and implemented BrainGazer, a system which integrates visualization techniques for volume data acquired through confocal microscopy as well as annotated anatomical structures with an intuitive approach for accessing the available information. We focus on the ability to visually query the data based on semantic as well as spatial relationships. Additionally, we present visualization techniques for the concurrent depiction of neurobiological volume data and geometric objects which aim to reduce visual clutter. The described system is the result of an ongoing interdisciplinary collaboration between neurobiologists and visualization researchers.

Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1.

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action is still incompletely understood. In the nematode Caenorhabditis elegans, 131 of 1090 somatic cells undergo programmed cell death during development. Transgenic expression of human Bcl-2 reduced cell death during nematode development, and partially complemented mutation of ced-9, indicating that Bcl-2 can functionally interact with the nematode cell death machinery. Identification of the nematode target(s) of Bcl-2 inhibition would help clarify the mechanism by which Bcl-2 suppresses apoptosis in mammalian cells. Exploiting yeast-based systems and biochemical assays, we analysed the ability of Bcl-2 to interact with and regulate the activity of nematode apoptosis proteins. Unlike CED-9, Bcl-2 could not directly associate with the caspase-activating adaptor protein CED-4, nor could it inhibit CED-4-dependent yeast death. By contrast, Bcl-2 could bind the C. elegans pro-apoptotic BH3-only Bcl-2 family member EGL-1. These data prompt us to hypothesise that Bcl-2 might suppress nematode cell death by preventing EGL-1 from antagonising CED-9, rather than by inhibiting CED-4.

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma.

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Sexual behaviour: do a few dead neurons make the difference?

Why do males and females behave so differently? Sexually dimorphic neural circuitry has just been found in parts of the fly's brain thought to control mating behaviour. Might this explain why males and females have such distinct sexual behaviours?