The readily releasable pool (RRP) of vesicles is a core concept in studies of presynaptic function. However, operating principles lack consensus definition and the utility for quantitative analysis has been questioned. Here we confirm that RRPs at calyces of Held from 14 to 21 day old mice have a fixed capacity for storing vesicles that is not modulated by Ca2+. Discrepancies with previous studies are explained by a dynamic flow-through pool, established during heavy use, containing vesicles that are released with low probability despite being immediately releasable. Quantitative analysis ruled out a posteriori explanations for the vesicles with low release probability, such as Ca2+-channel inactivation, and established unexpected boundary conditions for remaining alternatives. Vesicles in the flow-through pool could be incompletely primed, in which case the full sequence of priming steps downstream of recruitment to the RRP would have an average unitary rate of at least 9/s during heavy use. Alternatively, vesicles with low and high release probability could be recruited to distinct types of release sites; in this case the timing of recruitment would be similar at the two types, and the downstream transition from recruited to fully primed would be much faster. In either case, further analysis showed that activity accelerates the upstream step where vesicles are initially recruited to the RRP. Overall, our results show that the RRP can be well defined in the mathematical sense, and support the concept that the defining mechanism is a stable group of autonomous release sites.

Study DesignProspective cohort study.PurposeTo compare intraoperative parameters, radiation exposure, and pedicle screw perforation rate in navigation-guided versus non-navigated fluoroscopy-assisted minimal invasive transforaminal lumbar interbody fusion (MIS TLIF).Overview of LiteratureThe poor reliability of fluoroscopy-guided instrumentation and growing concerns about radiation exposure have led to the development of navigation-guided instrumentation techniques in MIS TLIF. The literature evaluating the efficacy of navigation-guided MIS TLIF is scant.MethodsEighty-seven patients underwent navigation- or fluoroscopy-guided MIS TLIF for symptomatic lumbar/lumbosacral spondylolisthesis. Demographics, intraoperative parameters (surgical time, blood loss), and radiation exposure (sec/mGy/Gy.cm2 noted from C-arm for comparison only) were recorded. Computed tomography was performed in patients in the navigation and non-navigation groups at postoperative 12 months and reviewed by an independent observer to assess the accuracy of screw placement, perforation incidence, location, grade (Mirza), and critical versus non-critical neurological implications.ResultsTwenty-seven patients (male/female, 11/16; L4–L5/L5–S1, 9/18) were operated with navigation-guided MIS TLIF, whereas 60 (male/female, 25/35; L4–L5/L5–S1, 26/34) with conventional fluoroscopy-guided MIS TILF. The use of navigation resulted in reduced fluoroscopy usage (dose area product, 0.47 Gy.cm2 versus 2.93 Gy.cm2), radiation exposure (1.68 mGy versus 10.97 mGy), and fluoroscopy time (46.5 seconds versus 119.08 seconds), with p-values of <0.001. Furthermore, 96.29% (104/108) of pedicle screws in the navigation group were accurately placed (grade 0) (4 breaches, all grade I) compared with 91.67% (220/240) in the non-navigation group (20 breaches, 16 grade I+4 grade II; p=0.114). None of the breaches resulted in a corresponding neurological deficit or required revision.ConclusionsNavigation guidance in MIS TLIF reduced radiation exposure, but the perforation status was not statistically different than that for the fluoroscopy-based technique. Thus, navigation in nondeformity cases is useful for significantly reducing the radiation exposure, but its ability to reduce pedicle screw perforation in nondeformity cases remains to be proven.

Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs’ ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.

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Key points This study characterizes the mechanisms underlying defects in synaptic transmission when dynamin‐related protein 1 (DRP1) is genetically eliminated. Viral‐mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of the synaptic vesicle recycling pool and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing the intracellular calcium buffering capacity with EGTA‐AM, implying close coupling of Ca2+ channels to synaptic vesicles was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not reverse the synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Abstract Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin‐related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of Cre recombinase in ventral cochlear neurons of floxed‐DRP1 mice generated a presynaptic‐specific DRP1 knockout (DRP1‐preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggested SV clusters and mitochondrial content were disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage‐clamp recordings, we found that DRP1‐preKO synapses had larger evoked responses at low frequency stimulation. DRP1‐preKO synapses also had profoundly altered short‐term plasticity, due to defects in SV recycling. Readily releasable pool size, estimated with high‐frequency trains, was dramatically reduced in DRP1‐preKO synapses, suggesting an important role for DRP1 in maintenance of release‐competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1‐preKO synapses. Synaptic transmission defects could be partially rescued with EGTA‐AM, indicating close coupling of Ca2+ channels to SV distance normally found in mature terminals may be compromised by DRP1‐preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation.

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Cortical gamma oscillations are believed to be involved in mental processes which are disturbed in schizophrenia. For example, the magnitudes of sensory-evoked oscillations, as measured by auditory steady-state responses (ASSRs) at 40 Hz, are robustly diminished, whereas the baseline gamma power is enhanced in schizophrenia. Such dual gamma oscillation abnormalities are also present in a mouse model of N-methyl-D-aspartate receptor hypofunction (Ppp1r2cre/Grin1 knockout mice). However, it is unclear whether the abnormal gamma oscillations are associated with dysfunction in schizophrenia. We found that glycogen synthase kinase-3 (GSK3) is overactivated in corticolimbic parvalbumin-positive GABAergic interneurons in Grin1 mutant mice. Here we addressed whether GSK3β inhibition reverses both abnormal gamma oscillations and behavioral deficits with high correlation by pharmacological and genetic approach. We demonstrated that the paralog selective-GSK3β inhibitor, but not GSK3α inhibitor, normalizes the diminished ASSRs, excessive baseline gamma power, and deficits in spatial working memory and prepulse inhibition (PPI) of acoustic startle in Grin1 mutant mice. Cell-type specific GSK3B knockdown, but not GSK3A knockdown, also reversed abnormal gamma oscillations and behavioral deficits. Moreover, GSK3B knockdown, but not GSK3A knockdown, reverses the mutants’ in vivo spike synchrony deficits. Finally, ex vivo patch-clamp recording from pairs of neighboring cortical pyramidal neurons showed a reduction of synchronous spontaneous inhibitory-postsynaptic-current events in mutants, which was reversed by GSK3β inhibition genetically and pharmacologically. Together, GSK3β inhibition in corticolimbic interneurons ameliorates the deficits in spatial working memory and PPI, presumably by restoration of synchronous GABA release, synchronous spike firing, and evoked-gamma power increase with lowered baseline power.

The present study was conducted to evaluate the clinico-physiological effects of epidural anaesthesia using lignocaine alone and in combination with ketamine in cow calves. Eight clinically healthy non-descript, stall-fed male cow calves aged 7 to 8 months and weighing 55 to 65 kg were used in the study. Animals were divided in two groups of four animals each. In (group A) lignocaine @ 0.5mg/kg body wt. and in (group B) lignocaine @ 0.5mg/kg body wt with ketamine @ 2 mg/kg body wt. was administered at first inter coccygeal epidural space. Clinical observations such as, onset, depth of analgesia, area of desensitization, motor incoordination, salivation, heart-rate, respiration rate and rectal temperature O ( F) were recorded before and at 5, 10, 20, 30, 45, 60, 90, 120, 150,180 and 240 minutes after injection of drug(s). Ruminal movements were recorded at every 30 minutes after the injection of drug(s) upto complete recovery and 24 hours after the injection. The onset of analgesia in group B was significantly shorter as compared to group A. Group B animals induced deeper analgesia as compared to group A. In group A animals decrease in heart rate was recorded whereas in group B animals heart rate was increased. In group A, decrease in RR was observed.