Optogenetic Tractography for anatomo-functional characterization of cortico-subcortical neural circuits in non-human primates.

Dissecting neural circuitry in non-human primates (NHP) is crucial to identify potential neuromodulation anatomical targets for the treatment of pharmacoresistant neuropsychiatric diseases by electrical neuromodulation. How targets of deep brain stimulation (DBS) and cortical targets of transcranial magnetic stimulation (TMS) compare and might complement one another is an important question. Combining optogenetics and tractography may enable anatomo-functional characterization of large brain cortico-subcortical neural pathways. For the proof-of-concept this approach was used in the NHP brain to characterize the motor cortico-subthalamic pathway (m_CSP) which might be involved in DBS action mechanism in Parkinson's disease (PD). Rabies-G-pseudotyped and Rabies-G-VSVg-pseudotyped EIAV lentiviral vectors encoding the opsin ChR2 gene were stereotaxically injected into the subthalamic nucleus (STN) and were retrogradely transported to the layer of the motor cortex projecting to STN. A precise anatomical mapping of this pathway was then performed using histology-guided high angular resolution MRI tractography guiding accurately cortical photostimulation of m_CSP origins. Photoexcitation of m_CSP axon terminals or m_CSP cortical origins modified the spikes distribution for photosensitive STN neurons firing rate in non-equivalent ways. Optogenetic tractography might help design preclinical neuromodulation studies in NHP models of neuropsychiatric disease choosing the most appropriate target for the tested hypothesis.

Action potential-evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones.

Axonal [Ca2+] transients evoked by action potential (AP) propagation were studied by monitoring the fluorescence of the high-affinity calcium-sensitive dye Oregon Green 488 BAPTA-1, introduced through whole-cell recording pipettes in the molecular layer of interneurones from cerebellar slices of young rats. The spatiotemporal profile of Ca2+-dependent fluorescence changes was analysed in well-focused axonal stretches a few tens of micrometres long. AP-evoked Ca2+ signals were heterogeneously distributed along axons, with the largest and fastest responses appearing in hot spots on average approximately 5 microm apart. The spatial distribution of fluorescence responses was independent of the position of the focal plane, uncorrelated to basal dye fluorescence, and independent of dye concentration. Recordings using the low-affinity dye mag-fura-2 and a Cs+-based intracellular solution revealed a similar pattern of hot spots in response to depolarisation, ruling out measurement artefacts or possible effects of inhomogeneous dye distribution in the generation of hot spots. Fluorescence responses to a short train of APs in hot spots decreased by 41-76 % after bath perfusion of omega-conotoxin MVIIC (5-6 microM), and by 17-65 % after application of omega-agatoxin IVA (500 nM). omega-Conotoxin GVIA (1 microM) had a variable, small effect (0-31 % inhibition), and nimodipine (5 microM) had none. Somatically recorded voltage-gated currents during depolarising pulses were unaffected in all cases. These data indicate that P/Q-type Ca2+ channels, and to a lesser extent N-type channels, are responsible for a large fraction of the [Ca2+] rise in axonalhot spots. [Ca2+] responses never failed during low-frequency (<= 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sites. Axonal branching points coincided with a hot spot in approximately 50 % of the cases. The spacing of presynaptic varicosities, as determined by a morphological analysis of Neurobiotin-filled axons, was approximately 10 times larger than the one measured for hot spots. The latter is comparable to the spacing reported for varicosities in mature animals. We discuss the nature of hot spots, considering as the most parsimonious explanation that they represent functional clusters of voltage-dependent Ca2+ channels, and possibly other [Ca2+] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.

Somatic recording of GABAergic autoreceptor current in cerebellar stellate and basket cells.

Patch-clamp recordings were performed from stellate and basket cells in rat cerebellar slices. Under somatic voltage clamp, short depolarizing pulses were applied to elicit action potentials in the axon. After the action potential, a bicuculline- and Cd2+-sensitive current transient was observed. A similar response was obtained when eliciting axonal firing by extracellular stimulation. With an isotonic internal Cl- solution, the peak amplitude of this current varied linearly with the holding potential, yielding an extrapolated reversal potential of -20 to 0 mV. Unlike synaptic or autaptic GABAergic currents obtained in the same preparation, the current transient had a slow rise-time and a low variability between trials. This current was blocked when 10 mM BAPTA was included in the recording solution. In some experiments, the current transient elicited axonal action potentials. The current transient was reliably observed in animals aged 12-15 d, with a mean amplitude of 82 pA at -70 mV, but was small and rare in the age group 29-49 d. Numerical simulations could account for all properties of the current transient by assuming that an action potential activates a distributed GABAergic conductance in the axon. The actual conductance is probably restricted to release sites, with an estimated mean presynaptic current response of 10 pA per site (-70 mV, age 12-15 d). We conclude that in developing rats, stellate and basket cell axons have a high density of GABAergic autoreceptors and that a sizable fraction of the corresponding current can be measured from the soma.

Autaptic inhibitory currents recorded from interneurones in rat cerebellar slices.

1. While the presence of autapses in the brain is indicated by a large body of morphological evidence, the functional role of these structures has remained unclear. To probe for autaptic currents, we have recorded current responses following short somatic depolarizing pulses in Cl--loaded interneurones (stellate and basket cells) from rat cerebellar slices (animals aged 27-39 days). 2. In approximately 20 % of the recordings, fluctuating inward current transients were obtained with a latency of 1.15-2.45 ms (measured from the peak of the depolarization-induced Na+ current; n = 10). 3. These transients were blocked by bicuculline and were sensitive to the extracellular Ca2+ concentration. 4. Assuming low release probability, as suggested by the high failure rate (0.65-0.92, n = 10), quantal sizes ranging from 21 to 178 pA (-70 mV; n = 10) were calculated from a variance analysis of autaptic current amplitudes. 5. We conclude that approximately 20 % of interneurones have a functional autapse. Autaptic currents may inhibit firing of interneurones during high frequency discharges.

Developmental regulation of basket/stellate cell-->Purkinje cell synapses in the cerebellum.

We used paired recordings to study the development of synaptic transmission between inhibitory interneurons of the molecular layer and Purkinje cells in the cerebellar cortex of the rat. The electrophysiological data were combined with a morphological study of the recorded cells using biocytin or Lucifer yellow staining. Thirty-one interneuron-Purkinje cell pairs were obtained, and 11 of them were recovered morphologically. The age of the rats ranged from 11 to 31 d after birth. During this period synaptic maturation resulted in an 11-fold decrease in the average current evoked in a Purkinje cell by a spike in a presynaptic interneuron. Unitary IPSCs in younger animals exhibited paired-pulse depression, whereas paired-pulse facilitation was found in more mature animals. These data suggest that reduction in transmitter release probability contributed to the developmental decrease of unitary IPSCs. However, additional mechanisms at both presynaptic and postsynaptic loci should also be considered. The decrease of the average synaptic current evoked in a Purkinje cell by an action potential in a single interneuron suggests that as development proceeds interneuron activities must be coordinated to inhibit efficiently Purkinje cells.

Monte Carlo simulation of fast excitatory synaptic transmission at a hippocampal synapse.

1. A simulation of fast excitatory synaptic transmission at a hippocampal synapse is presented. Individual neurotransmitter molecules are followed as they diffuse through the synaptic cleft and interact with the postsynaptic receptors. The ability of the model to reproduce published results of patch-clamp experiments on CA3 pyramidal cells is illustrated; parameters of the model that affect the time course and variability of the excitatory postsynaptic current (EPSC) are then investigated. 2. To simulate an EPSC, we release 4,000 neurotransmitter molecules simultaneously from a point source centered 15 nm above a rectangular grid of 14 x 14 postsynaptic receptors. The simulated EPSC at room temperature has a 10-90% rise time of 0.28 ms and a peak open probability of 0.27, and decays with a time constant of 2.33 ms, comparing well with values in the literature. 3. To simulate changes in temperature, we use a 10 degrees temperature coefficient (Q10) for diffusion of 1.3 and apply a Q10 of 3.0 to all the rate constants of the kinetic scheme. At 37 degrees C, the 10-90 rise time is 0.07 ms, the peak open probability is 0.56, and the decay time constant is 0.70 ms. The coefficient of variation (CV) at the peak of the EPSC is 9.4% at room temperature; at 37 degrees C, the CV at the peak drops to 6.6%. 4. We use the diffusion coefficient of glutamine, 7.6 x 10(-6) cm2/s, to model the random movement of glutamate molecules in the synaptic cleft. Slower rates of diffusion increase the peak response and slow the time course of decay of the EPSC. 5. Random variations in release site position have little effect on the time course of the average EPSC or on the CV of the peak response. We simulate a dose-response curve for the effects of releasing between 100 and 7,500 neurotransmitter molecules per vesicle. The half-maximal response occurs for 1,740 molecules. For a simulation with 100 postsynaptic receptors and a diffusion coefficient of 2.0 x 10(-6) cm2/s, 4,000 molecules approaches a saturating dose. 6. Changes to the width of the synaptic cleft, or to the number and spacing of the postsynaptic receptors, have marked effects on the peak height of the simulated EPSC. 7. We extend the model to include a spherical vesicle (50 nm diam) connected to the synaptic cleft by a cylindrical pore 15 nm long. Neurotransmitter molecules are randomly distributed within the vesicle and allowed to diffuse into the synaptic cleft through the pore, which opens to its full diameter in one time step. We find that the pore must open to a diameter of > or = 7 nm within 1 microsecond in order to match the time courses of EPSCs in the literature.

A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release.

Previous studies have shown that 4-aminopyridine (4AP) induced Ca-influx effects the release of glutamate from nerve terminals (synaptosomes) isolated from rat cerebral cortex. We now show that the Ca-dependent component of this release is potentiated by preincubation of the synaptosomes with the immunosuppressant, FK506, an inhibitor of protein phosphatase-2B (calcineurin). FK506 did not inhibit the Ca-independent release of glutamate from a cytosolic pool. Examination of the effect of FK506 on the influx of Ca elicited by 4AP indicated that inhibition of calcineurin activity resulted in an increase of voltage-dependent Ca-influx. Based on these results, we suggest that protein dephosphorylation effected by calcineurin may suppress voltage-dependent Ca-channel activity and in so doing inhibits evoked glutamate release. Activation of calcineurin produced by initial Ca-entry may represent a negative feedback to limit the activity of Ca-channels coupled to the release of glutamate.