Gamma System Research Articles

Neural computational modeling reveals a major role of corticospinal gating of central oscillations in the generation of essential tremor.

Essential tremor, also referred to as familial tremor, is an autosomal dominant genetic disease and the most common movement disorder. It typically involves a postural and motor tremor of the hands, head or other part of the body. Essential tremor is driven by a central oscillation signal in the brain. However, the corticospinal mechanisms involved in the generation of essential tremor are unclear. Therefore, in this study, we used a neural computational model that includes both monosynaptic and multisynaptic corticospinal pathways interacting with a propriospinal neuronal network. A virtual arm model is driven by the central oscillation signal to simulate tremor activity behavior. Cortical descending commands are classified as alpha or gamma through monosynaptic or multisynaptic corticospinal pathways, which converge respectively on alpha or gamma motoneurons in the spinal cord. Several scenarios are evaluated based on the central oscillation signal passing down to the spinal motoneurons via each descending pathway. The simulated behaviors are compared with clinical essential tremor characteristics to identify the corticospinal pathways responsible for transmitting the central oscillation signal. A propriospinal neuron with strong cortical inhibition performs a gating function in the generation of essential tremor. Our results indicate that the propriospinal neuronal network is essential for relaying the central oscillation signal and the production of essential tremor.

Effect of streptozotocin-induced diabetes on motoneurons and muscle spindles in rats

This study examined the alterations in the number and size of motoneurons innervating the medial gastrocnemius (MG) and biceps femoris (BF) motor nuclei in diabetic rats (12 or 22 weeks after injection of streptozotocin) and age-matched controls using retrograde labeling technique. Additionally, morphological alterations of muscle spindles in BF and MG muscles were tested. Significantly fewer labeled MG motoneurons were found in 12- and 22-week diabetic rats as compared with age-matched control animals. In contrast, the number of BF motoneurons was preserved in each group. Compared to control animals, the ratio of larger motoneurons of MG and BF muscle were decreased at 12 weeks, and smaller MG motoneurons were drastically decreased at 22 weeks. Moreover, MG muscle spindle showed reduction of its number and increase of intrafusal muscle fibers; however, BF muscle spindles showed little or no difference from control animals. We conclude that there is an early loss of alpha motoneurons for both MG and BF muscles followed by a later loss of gamma motoneurons in MG muscle in diabetic animals. Moreover, loss of gamma motoneuron might induce atrophy of MG muscle spindles.

ID 57 – End plate spikes or fusimotor spikes

Objective End plate spikes (EPSs) are often recorded in EMG studies. They are usually accompanied by miniature end plate potentials (MEPPs). The origin of EPSs is conjectured to be irritation of a terminal motor nerve twig by the needle electrode. The EPSs are then recorded postsynaptically from the muscle fibre. I suggest another explanation for EPSs. Methods EPSs were recorded from healthy voluntary persons using one or several needle electrodes in parallel with the muscle fibres. We also used local anesthesia of the nerve outside the given muscle. Results EPSs were observed to be usually local, without propagation to other needle electrodes in parallel with the muscle fibres. Some EPSs propagated for a short distance and, rarely, we observed EPSs propagating like motor unit potentials. Local anesthesia suppressed EPSs completely but 80% of the M-response was spared. Passive stretch of the muscle activated EPSs. Conclusions These observations point out that EPSs represent action potentals of intrafusal motor units. Local EPSs reflect junction potentials of nuclear bag fibres and shortly propagating action potentials action potentials of nuclear chain fibres. MUP-like EPSs represent potentals of beta motor units. Stretch activates II-afferents which synapse to gamma and beta motor neurones. Key message EPSs are intrafusal.

Modulation of gamma and alpha spinal motor neurons activity by trans-spinal direct current stimulation: effects on reflexive actions and locomotor activity.

Spontaneous and evoked spinal activities interact to set the characteristics of emergent motor responses. Gamma motor neurons have feedforward and feedback functions in motor control, which are crucial for transforming motor commands into action. Meanwhile, the intrinsic excitability and functional connectivity of alpha motor neurons determine the accuracy of actions. In this study, we investigated the effects of trans-spinal direct current stimulation (tsDCS) on spontaneous and cortically evoked activity of well-isolated single units of gamma and alpha motor neurons in mice. We also investigated the effects of tsDCS on reflexive and locomotor actions. In general, motor neurons showed increased responses to cathodal tsDCS (c-tsDCS) and decreased responses to anodal tsDCS (a-tsDCS). These effects were observed for cortically evoked discharges and spontaneous firing rates of gamma motor neurons, cortically evoked discharges of larger alpha motor neurons, and spontaneous firing rates of smaller alpha motor neurons. An exception was that spontaneous firing rates of larger alpha motor neurons showed the opposite pattern of reduction by c-tsDCS and increase by a-tsDCS. Reflexive and voluntary behavior were also increased by c-tsDCS and reduced by a-tsDCS. Specifically, the amplitude and duration of crossed and tail pinch reflexes in decerebrate animals and the quality of ground and treadmill walking patterns in healthy awake animals showed this pattern. These polarity-specific changes in behavior could be attributed to polarity-mediated modulation of alpha and gamma motor neuron activity and spinal circuitry. The results reveal an important principle: effects of tsDCS on spinal motor neurons depend on current polarity and cell size.

High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord.

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases.

Selective loss of alpha motor neurons with sparing of gamma motor neurons and spinal cord cholinergic neurons in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterised primarily by loss of lower motor neurons from the ventral grey horn of the spinal cord and proximal muscle atrophy. Recent experiments utilising mouse models of SMA have demonstrated that not all motor neurons are equally susceptible to the disease, revealing that other populations of neurons can also be affected. Here, we have extended investigations of selective vulnerability of neuronal populations in the spinal cord of SMA mice to include comparative assessments of alpha motor neuron (α-MN) and gamma motor neuron (γ-MN) pools, as well as other populations of cholinergic neurons. Immunohistochemical analyses of late-symptomatic SMA mouse spinal cord revealed that numbers of α-MNs were significantly reduced at all levels of the spinal cord compared with controls, whereas numbers of γ-MNs remained stable. Likewise, the average size of α-MN cell somata was decreased in SMA mice with no change occurring in γ-MNs. Evaluation of other pools of spinal cord cholinergic neurons revealed that pre-ganglionic sympathetic neurons, central canal cluster interneurons, partition interneurons and preganglionic autonomic dorsal commissural nucleus neuron numbers all remained unaffected in SMA mice. Taken together, these findings indicate that α-MNs are uniquely vulnerable among cholinergic neuron populations in the SMA mouse spinal cord, with γ-MNs and other cholinergic neuronal populations being largely spared.

On the distribution of information from muscle spindles in the spinal cord; how much does it depend on random factors?

Information forwarded by individual muscle spindles is modulated by the dynamic and static gamma motoneurons in a differentiated way, depending on the coupling between the fusimotor neurons and the various intrafusal muscle fibres. Further modulation of this information at the level of spinal neurons is also differentiated because connections between individual muscle spindles and their spinal target cells are quite variable. This review illustrates this variability with respect to the spinal trajectory of muscle spindle primary afferents and the distribution of their synaptic contacts on motoneurons and other spinal neurons. It also discusses some of the consequences of this variability for the processing of information from proprioceptors.

Principles and mechanisms of regeneration in the mouse model for wound-induced hair follicle neogenesis.

Wound‐induced hair follicle neogenesis (WIHN) describes a regenerative phenomenon in adult mammalian skin wherein fully functional hair follicles regenerate de novo in the center of large excisional wounds. Originally described in rats, rabbits, sheep, and humans in 1940−1960, the WIHN phenomenon was reinvestigated in mice only recently. The process of de novo hair regeneration largely duplicates the morphological and signaling features of normal embryonic hair development. Similar to hair development, WIHN critically depends on the activation of canonical WNT signaling. However, unlike hair development, WNT activation in WIHN is dependent on fibroblast growth factor 9 signaling generated by the immune system's γδ T cells. The cellular bases of WIHN remain to be fully characterized; however, the available evidence leaves open the possibility for a blastema‐like mechanism wherein epidermal and/or dermal wound cells undergo epigenetic reprogramming toward a more plastic, embryonic‐like state. De novo hair follicles do not regenerate from preexisting hair‐fated bulge stem cells. This suggests that hair neogenesis is not driven by preexisting lineage‐restricted progenitors, as is the case for amputation‐induced mouse digit tip regeneration, but rather may require a blastema‐like mechanism. The WIHN model is characterized by several intriguing features, which await further explanation. These include (1) the minimum wound size requirement for activating neogenesis, (2) the restriction of hair neogenesis to the wound's center, and (3) imperfect patterning outcomes, both in terms of neogenic hair positioning within the wound and in terms of their orientation. Future enquiries into the WIHN process, made possible by a wide array of available skin‐specific genetic tools, will undoubtedly expand our understanding of the regeneration mechanisms in adult mammals.

Muscle spindle and fusimotor activity in locomotion.

Mammals may exhibit different forms of locomotion even within a species. A particular form of locomotion (e.g. walk, run, bound) appears to be selected by supraspinal commands, but the precise pattern, i.e. phasing of limbs and muscles, is generated within the spinal cord by so-called central pattern generators. Peripheral sense organs, particularly the muscle spindle, play a crucial role in modulating the central pattern generator output. In turn, the feedback from muscle spindles is itself modulated by static and dynamic fusimotor (gamma) neurons. The activity of muscle spindle afferents and fusimotor neurons during locomotion in the cat is reviewed here. There is evidence for some alpha-gamma co-activation during locomotion involving static gamma motoneurons. However, both static and dynamic gamma motoneurons show patterns of modulation that are distinct from alpha motoneuron activity. It has been proposed that static gamma activity may drive muscle spindle secondary endings to signal the intended movement to the central nervous system. Dynamic gamma motoneuron drive appears to prime muscle spindle primary endings to signal transitions in phase of the locomotor cycle. These findings come largely from reduced animal preparations (decerebrate) and require confirmation in freely moving intact animals.

Speech encoding by coupled cortical theta and gamma oscillations.

Many environmental stimuli present a quasi-rhythmic structure at different timescales that the brain needs to decompose and integrate. Cortical oscillations have been proposed as instruments of sensory de-multiplexing, i.e., the parallel processing of different frequency streams in sensory signals. Yet their causal role in such a process has never been demonstrated. Here, we used a neural microcircuit model to address whether coupled theta-gamma oscillations, as observed in human auditory cortex, could underpin the multiscale sensory analysis of speech. We show that, in continuous speech, theta oscillations can flexibly track the syllabic rhythm and temporally organize the phoneme-level response of gamma neurons into a code that enables syllable identification. The tracking of slow speech fluctuations by theta oscillations, and its coupling to gamma-spiking activity both appeared as critical features for accurate speech encoding. These results demonstrate that cortical oscillations can be a key instrument of speech de-multiplexing, parsing, and encoding.

Exploring the high-dimensional structure of muscle redundancy via subject-specific and generic musculoskeletal models

Subject-specific and generic musculoskeletal models are the computational instantiation of hypotheses, and stochastic techniques help explore their validity. We present two such examples to explore the hypothesis of muscle redundancy. The first addresses the effect of anatomical variability on static force capabilities for three individual cat hindlimbs, each with seven kinematic degrees of freedom (DoFs) and 31 muscles. We present novel methods to characterize the structure of the 31-dimensional set of feasible muscle activations for static force production in every 3-D direction. We find that task requirements strongly define the set of feasible muscle activations and limb forces, with few differences comparing individual vs. species-average results. Moreover, muscle activity is not smoothly distributed across 3-D directions. The second example explores parameter uncertainty during a flying disc throwing motion by using a generic human arm with five DoFs and 17 muscles to predict muscle fiber velocities. We show that the measured joint kinematics fully constrain the eccentric and concentric fiber velocities of all muscles via their moment arms. Thus muscle activation for limb movements is likely not redundant: there is little, if any, latitude in synchronizing alpha–gamma motoneuron excitation–inhibition for muscles to adhere to the time-critical fiber velocities dictated by joint kinematics. Importantly, several muscles inevitably exhibit fiber velocities higher than thought tenable, even for conservative throwing speeds. These techniques and results, respectively, enable and compel us to continue to revise the classical notion of muscle redundancy for increasingly more realistic models and tasks.

Astrocyte-encoded positional cues maintain sensorimotor circuit integrity

SUMMARYAstrocytes, the most abundant cells in the central nervous system, promote synapse formation and help refine neural connectivity. Although they are allocated to spatially distinct regional domains during development, it is unknown whether region-restricted astrocytes are functionally heterogeneous. Here we show that postnatal spinal cord astrocytes express several region-specific genes, and that ventral astrocyte-encoded Semaphorin3a (Sema3a) is required for proper motor neuron and sensory neuron circuit organization. Loss of astrocyte-encoded Sema3a led to dysregulated α–motor neuron axon initial segment orientation, markedly abnormal synaptic inputs, and selective death of α–but not of adjacent γ–motor neurons. Additionally, a subset of TrkA+ sensory afferents projected to ectopic ventral positions. These findings demonstrate that stable maintenance of a positional cue by developing astrocytes influences multiple aspects of sensorimotor circuit formation. More generally, they suggest that regional astrocyte heterogeneity may help to coordinate postnatal neural circuit refinement.

Proprioception in the cerebellum.

Proprioception is the ability to interpret our musculo-skeletal state (e.g., position and movement) by processing information originating from our own body. While it is generally accepted that passive proprioception (i.e., proprioception in the absence of muscle contraction) is dependent only on the processing of peripheral inputs, the precise nature of the processes constituting active proprioception (i.e., proprioception with muscle contraction) is still not clear (Proske and Gandevia, 2012). Central to this knowledge gap is the difficulty in accurately determining the processes responsible for the improvement of proprioception in active compared to passive movements (e.g., Fuentes and Bastian, 2010). This improvement is assumed to potentially result from [1] enhanced peripheral muscle information (through gamma motoneurons of the muscle spindles), [2] the direct transmission of a copy of motor commands (i.e., efference copy) from motor to sensory processing areas, and/or [3] the involvement of predictive models through the cerebellum (Wolpert and Miall, 1996; Bhanpuri et al., 2012; Proske and Gandevia, 2012). Here, based on the results of Bhanpuri et al. (2013), we propose that cerebellar predictive models can fully account for the improvement of proprioception in active movements. We also describe the cellular arrangement that may underlie the involvement of predictive models in proprioception. In their recent study, Bhanpuri et al. (2013) tested proprioception in cerebellar patients and well matched controls. They measured perceptual thresholds of the dominant arm using an exoskeleton robot system in three proprioceptive tasks: passive, active (active-simple), and active with a complex pattern of resistive torque during forearm displacement making this displacement unpredictable (active-complex). Based on Weber fractions, controls demonstrated better proprioception in the active-simple task compared to the passive (p 0.38). In contrast, no differences were found between any of the tasks in cerebellar patients (all p > 0.28), who performed worse than controls in the active-simple task (p < 0.03). The similar performance observed in the passive and active-complex task in controls reveals that the proprioceptive benefit usually observed in active compared to passive conditions is neither related to an enhancement of information from the peripheral muscle [1] nor to the direct transmission of an efference copy from motor to sensory areas [2]. Indeed, these two processes may have been operating in the active-complex task but did not improve proprioceptive performance compared to the passive task. Furthermore, the absence of improvement in the active-simple task compared to the passive task in cerebellar patients demonstrates that the improved proprioception observed in the active-simple task in controls can be fully accounted for by a process located in the cerebro-cerebellar loop. Finally, cerebellar patients demonstrated a lower performance level than controls in the active-simple (predictable) task but not in the active-complex (non-predictable) task. Therefore the process that is most likely responsible for the improvement in proprioception is predictive modeling, which is thought to be supported by the cerebellum [3]. A recent study in mice demonstrates that individual granule cells in the cerebellum can mix proprioceptive afferents from the spinocerebellar tract and efference copy from the cerebral cortex (Huang et al., 2013). This multimodal arrangement provides the anatomical basis for converting motor corollary discharges into sensory coordinates. Indeed, granule cells can generate action potentials in response to a single input (Rancz et al., 2007). Therefore, sensory and motor-related inputs can potentially substitute for one another to fire a granule cell. The nature of the granule cells' output is still unknown. However, studies testing experimental phantom limbs by blocking peripheral nerves revealed conscious sensations of limb movement in the absence of any sensory input when subjects attempted to move (e.g., Gandevia et al., 2006). This result clearly supports a conversion of the efference copy from motor to sensory coordinates making proprioceptive prediction possible. So far, the literature has emphasized the role of predictive models in the control of movement where intended actions are compared with actual actions to generate error signals. However, results from Bhanpuri et al. (2013) as well as studies of phantom limbs demonstrate that the purpose of cerebellar processes is not only motor control but also proprioception per se.

Dopaminergic Modulation of cAMP Drives Nonlinear Plasticity across the Drosophila Mushroom Body Lobes

Activity of dopaminergic neurons is necessary and sufficient to evoke learning-related plasticity in neuronal networks that modulate learning. During olfactory classical conditioning, large subsets of dopaminergic neurons are activated, releasing dopamine across broad sets of postsynaptic neurons. It is unclear how such diffuse dopamine release generates the highly localized patterns of plasticity required for memory formation. Here we have mapped spatial patterns of dopaminergic modulation of intracellular signaling and plasticity in Drosophila mushroom body (MB) neurons, combining presynaptic thermogenetic stimulation of dopaminergic neurons with postsynaptic functional imaging in vivo. Stimulation of dopaminergic neurons generated increases in cyclic AMP (cAMP) across multiple spatial regions in the MB. However, odor presentation paired with stimulation of dopaminergic neurons evoked plasticity in Ca(2+) responses in discrete spatial patterns. These patterns of plasticity correlated with behavioral requirements for each set of MB neurons in aversive and appetitive conditioning. Finally, broad elevation of cAMP differentially facilitated responses in the gamma lobe, suggesting that it is more sensitive to elevations of cAMP and that it is recruited first into dopamine-dependent memory traces. These data suggest that the spatial pattern of learning-related plasticity is dependent on the postsynaptic neurons' sensitivity to cAMP signaling. This may represent a mechanism through which single-cycle conditioning allocates short-term memory to a specific subset of eligible neurons (gamma neurons).

Decreased Succinate Dehydrogenase Activity of Gamma and Alpha Motoneurons in Mouse Spinal Cords Following 13 Weeks of Exposure to Microgravity

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.

Sex Differences in the Number and Size of Motoneurons Innervating Rat Medial Gastrocnemius Muscle

The sex differences in the number and morphometric parameters of motoneurons in motor nuclei are poorly known. The aim of this study was to determine the differences in the number and size of alpha and gamma motoneurons of the medial gastrocnemius (MG) muscle in male and female Wistar rats. Retrogradely labelled cell bodies of motoneurons of 6 months old animals were studied following a bath of the proximal stump of the transected MG nerve in a horseradish peroxidase solution. The number and soma diameters of male and female MG motoneurons were determined from serial microscopic images of sections. The weight of the brain and spinal cord was on average 17% higher in males than in females. The mean number of motoneurons was 13% higher in males than in females and amounted to 94 and 83 motoneurons, respectively. In each case, the average soma diameters and cross-section areas of motoneurons in motor nucleus were distributed bimodally: motoneurons smaller than 27.5 μm in diameter were recognized as gamma and greater ones as alpha motoneurons. In males, the motor nucleus contained on the average 66 alpha motoneurons, whereas in females, 56 alpha motoneurons, that is the mean number of alpha motoneurons was 17% higher in males. Moreover, the soma diameters of gamma and alpha motoneurons were significantly bigger in males and the difference amounted 9 and 6%, respectively. It is concluded that the number as well as size of alpha and size of gamma motoneurons in the MG motor nucleus are greater in males.

Analytical Insights on Theta-Gamma Coupled Neural Oscillators

In this paper, we study the dynamics of a quadratic integrate-and-fire neuron, spiking in the gamma (30–100 Hz) range, coupled to a delta/theta frequency (1–8 Hz) neural oscillator. Using analytical and semianalytical methods, we were able to derive characteristic spiking times for the system in two distinct regimes (depending on parameter values): one regime where the gamma neuron is intrinsically oscillating in the absence of theta input, and a second one in which gamma spiking is directly gated by theta input, i.e., windows of gamma activity alternate with silence periods depending on the underlying theta phase. In the former case, we transform the equations such that the system becomes analogous to the Mathieu differential equation. By solving this equation, we can compute numerically the time to the first gamma spike, and then use singular perturbation theory to find successive spike times. On the other hand, in the excitable condition, we make direct use of singular perturbation theory to obtain an approximation of the time to first gamma spike, and then extend the result to calculate ensuing gamma spikes in a recursive fashion. We thereby give explicit formulas for the onset and offset of gamma spike burst during a theta cycle, and provide an estimation of the total number of spikes per theta cycle both for excitable and oscillator regimes.

Anatomically remote muscle contraction facilitates patellar tendon reflex reinforcement while mental activity does not: a within-participants experimental trial

BackgroundThe Jendrassik maneuver (JM) is a remote facilitation muscular contraction shown to affect amplitude and temporal components of the human stretch reflex. Conflicting theoretical models exist regarding the neurological mechanism related to its ability to reinforce reflex parameters. One mechanism involves the gamma motoneurons of the fusimotor system, which are subject to both physical and mental activity. A second mechanism describes reduced alpha motoneuron presynaptic inhibition, which is not subject to mental activity. In the current study, we determined if mental activity could be used to create a reflex facilitation comparable to a remote muscle contraction.MethodUsing a within-participants design, we investigated the relative effect of the JM and a successfully employed mental task (Stroop task) on the amplitude and temporal components of the patellar tendon reflex.ResultsWe found that the addition of mental activity had no influence on the patellar tendon reflex parameters measured, while the JM provided facilitation (increased reflex amplitude, decreased total reflex time).ConclusionThe findings from this study support the view that the mechanism for the JM is a reduction in presynaptic inhibition of alpha motoneurons as it is influenced by physical and not mental activity.

&lt;em&gt;Ex vivo&lt;/em&gt; Culturing of Whole, Developing &lt;em&gt;Drosophila&lt;/em&gt; Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab(1) has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)(2), was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine(3) causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically. A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly(4), and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern(5). Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches(6), by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1(7), and that is dependent on the activity of the ubiquitin-proteasome system(6). Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

&lt;em&gt;Ex vivo&lt;/em&gt; Culturing of Whole, Developing &lt;em&gt;Drosophila&lt;/em&gt; Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically. A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.