Jim Deuchars

Temporal and spatial properties of local circuits in neocortex

A large body of anatomical data has detailed many complexities of neocortical circuitry, and physiological studies have indicated some roles for this circuitry in the complex functions of the cortex. Until recently, however, we have little precise information about the spatio-temporal properties of synaptic connections between individual neocortical neurones. Studies of synaptic responses elicited in one neocortical neurone by action potentials in another, and parallel morphological studies that have identified these neurones and the synaptic connections between them, have now described these parameters for certain types of local circuit connection in the neocortex. Some of these studies confirmed previous observations and inferences, but others provided major surprises. Evidence indicates that the class of both the presynaptic and postsynaptic neurone together determine a wide range of synaptic properties, such as the type of postsynaptic receptors involved and the temporal pattern of transmitter release, so that each type of synapse displays unique properties. A role for retrograde diffusable messages in determining the temporal properties of these circuits is postulated.

Neuronal P2X7 Receptors Are Targeted to Presynaptic Terminals in the Central and Peripheral Nervous Systems

The ionotropic ATP receptor subunits P2X1–6 receptors play important roles in synaptic transmission, yet the P2X7receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X7 receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X7 receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X7 receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP; 30 μm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X7 receptor antagonists oxidized ATP (100 μm) and Brilliant Blue G (2 μm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X7 receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1–43 in isolated NMJ preparations destained after application of BzATP (30 μm). This BzATP evoked destaining is blocked by oxidized ATP (100 μm) and Brilliant Blue G (1 μm). This indicates that activation of the P2X7 receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

Single axon IPSPs elicited in pyramidal cells by three classes of interneurones in slices of rat neocortex.

1. Using dual intracellular recordings in slices of adult rat neocortex, twenty‐four IPSPs activated by single presynaptic interneurones were studied in simultaneously recorded pyramidal cells. Fast spiking interneurones inhibited one in four or five of their close pyramidal neighbours. No reciprocal connections were observed. After recordings neurones were filled with biocytin. 2. Interneurones that elicited IPSPs were classified as classical fast spiking (n = 10), as non‐classical fast spiking (n = 3, including one burst‐firing interneurone), as unclassified, or slow interneurones (n = 8), or as regular spiking interneurones (n = 3), i.e. interneurones whose electrophysiological characteristics were indistinguishable from those of pyramidal cells. 3. All of the seven classical fast spiking cells anatomically fully recovered had aspiny, beaded dendrites. Their partially myelinated axons ramified extensively, varying widely in shape and extent, but randomly selected labelled axon terminals typically innervated somata and large calibre dendrites on electron microscopic examination. One ‘autapse’ was demonstrated. One presumptive regular spiking interneurone axon made four somatic and five dendritic connections with unlabelled targets. 4. Full anatomical reconstructions of labelled classical fast spiking interneurones and their postsynaptic pyramids (n = 5) demonstrated one to five boutons per connection. The two recorded IPSPs that were fully reconstructed morphologically (3 and 5 terminals) were, however, amongst the smallest recorded (< 0.4 mV). Some connections may therefore involve larger numbers of contacts. 5. Single axon IPSPs were between 0.2 and 3.5 mV in average amplitude at ‐55 to ‐60 mV. Extrapolated reversal potentials were between ‐70 and ‐82 mV. IPSP time course correlated with the type of presynaptic interneurone, but not with IPSP latency, amplitude, reversal potential, or sensitivity to current injected at the soma. 6. Classical fast spiking interneurones elicited the fastest IPSPs (width at half‐amplitude 14.72 +/‐ 3.83 ms, n = 10) and unclassified, or slow interneurones the slowest (56.29 +/‐ 23.44 ms, n = 8). Regular spiking interneurone IPSPs had intermediate half‐widths (27.3 +/‐ 3.68 ms, n = 3). 7. Increasingly brief presynaptic interspike intervals increased the peak amplitude of, but not the area under, the summed IPSP. Only at interspike intervals between 10 and 20 ms did IPSP integrals exhibit paired pulse facilitation. Paired pulse depression was apparent at < 10 and 20‐60 ms. During longer spike trains, summing IPSPs decayed to a plateau potential that was relatively independent of firing rate (100‐250 Hz). Thereafter, the voltage response could increase again. 8. Summed IPSPs elicited by two to fifteen presynaptic spike trains decayed as, or more rapidly than, single‐spike IPSPs. Summed IPSPs elicited by > 20 spikes (> 150 Hz), however, resulted in an additional, more slowly decaying component (latency > 50 ms, duration > 200 ms). The possible involvement of GABAB receptors in this component is discussed. 9. It is suggested that three broad classes of interneurones may activate GABAA receptors on relatively proximal portions of neocortical pyramidal neurones. The different time courses of the IPSPs elicited by the three classes may reflect different types of postsynaptic receptor rather than dendritic location. An additional class, burst firing, spiny interneurones appear to activate GABAA receptors on more distal sites.

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus.

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7R) are largely unknown. Here we studied the effect of P2X7R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1–30 mm) and other ATP analogues elicited concentration‐dependent [3H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non‐selective P2‐receptor antagonist (3–30 µm), Brilliant blue G (1–100 nm) the P2X7‐selective antagonist and Zn2+ (0.1–30 µm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3–30 µm) and BzATP (0.6–6 µm) elicited concentration‐dependent [14C]glutamate efflux, and blockade of the kainate receptor‐mediated transmission by CNQX (10–100 µm) and gadolinium (100 µm), decreased ATP evoked [3H]GABA efflux. The Na+ channel blocker TTX (1 µm), low temperature (12°C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP‐induced [3H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation‐induced [3H]GABA efflux. In conclusion, P2X7Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.

CA1 pyramid-pyramid connections in rat hippocampus in vitro : dual intracellular recordings with biocytin filling

In adult rat hippocampus, simultaneous intracellular recordings from 989 pairs of CA1 pyramidal cells revealed nine monosynaptic, excitatory connections. Six of these pairs were sufficiently stable for electrophysiological analysis. Mean excitatory postsynaptic potential amplitude recorded at a postsynaptic membrane potential between -67 and -70 mV was 0.7 +/- 0.5 mV (0.17-1.5 mV), mean 10-90% rise time was 2.7 +/- 0.9 ms (1.5-3.8 ms) and mean width at half-amplitude was 16.8 +/- 4.1 ms (11.6-25 ms). Cells were labelled with biocytin and identified histologically. For one pair that was fully reconstructed morphologically, excitatory postsynaptic potential average amplitude was 1.5 mV, 10-90% rise time 2.8 ms and width at half-amplitude 11.6 ms (at -67 mV). In this pair, correlated light and electron microscopy revealed that the presynaptic axon formed two synaptic contacts with third-order basal dendrites of the postsynaptic pyramid, one with a dendritic spine, the other with a dendritic shaft. In the four pairs tested, postsynaptic depolarization increased excitatory postsynaptic potential amplitude and duration. In two, D-2-amino-5-phosphonovalerate (50 microM) reduced the amplitude and duration of the excitatory postsynaptic potential. The remainder of the excitatory postsynaptic potential now increased with postsynaptic hyperpolarization and was abolished by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (n = 1). Paired-pulse depression was evident in the four excitatory postsynaptic potentials tested. This depression decreased with increasing inter-spike interval. These results provide the first combined electrophysiological and morphological illustration of synaptic contacts between pyramidal neurons in the hippocampus and confirm that connections between CA1 pyramidal neurons are mediated by both N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors.

Adenoviral vector demonstrates that angiotensin II‐induced depression of the cardiac baroreflex is mediated by endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat

1 Angiotensin II (ANGII) acting on ANGII type 1 (AT1) receptors in the solitary tract nucleus (NTS) depresses the baroreflex. Since ANGII stimulates the release of nitric oxide (NO), we tested whether the ANGII‐mediated depression of the baroreflex in the NTS depended on NO release. 2 In a working heart‐brainstem preparation (WHBP) of rat NTS microinjection of either ANGII (500 fmol) or a NO donor (diethylamine nonoate, 500 pmol) both depressed baroreflex gain by ‐56 and ‐67 %, respectively (P < 0.01). In contrast, whilst ANGII potentiated the peripheral chemoreflex, the NO donor was without effect. 3 NTS microinjection of non‐selective NO synthase (NOS) inhibitors (l‐NAME; 50 pmol) or (l‐NMMA; 200 pmol) prevented the ANGII‐induced baroreflex attenuation (P > 0.1). In contrast, a neurone‐specific NOS inhibitor, TRIM (50 pmol), was without effect. 4 Using an adenoviral vector, a dominant negative mutant of endothelial NOS (TeNOS) was expressed bilaterally in the NTS. Expression of TeNOS affected neither baseline cardiovascular parameters nor baroreflex sensitivity. However, ANGII microinjected into the transfected region failed to affect the baroreflex. 5 Immunostaining revealed that eNOS‐positive neurones were more numerous than those labelled for AT1 receptors. Neurones double labelled for both AT1 receptors and eNOS comprised 23 ± 5.4 % of the eNOS‐positive cells and 57 ± 9.2 % of the AT1 receptor‐positive cells. Endothelial cells were also double labelled for eNOS and AT1 receptors. 6 We suggest that ANGII activates eNOS located in either neurones and/or endothelial cells to release NO, which acts selectively to depress the baroreflex.

Single axon excitatory postsynaptic potentials in neocortical interneurons exhibit pronounced paired pulse facilitation

In slices of adult rat somatosensory/motor cortex, paired recordings were made from pyramidal and non-pyramidal neurons. Single axon excitatory postsynaptic potentials evoked in the non-pyramidal neuron by action potentials in the pyramidal neuron were large and fast and demonstrated large fluctuations in amplitude, with coefficients of variation between 0.1 and 1.25. Excitatory postsynaptic potential amplitude distributions included a large number of apparent failures of transmission as well as some extremely large events. This contrasted dramatically with the relatively narrow distribution of amplitudes for pyramid-pyramid connections in neocortex. Excitatory postsynaptic potentials increased in amplitude with postsynaptic membrane hyperpolarization. Very small changes in the coefficient of variation when mean amplitudes increased substantially were consistent with the increase being due to a change in quantal amplitude. These excitatory postsynaptic potentials displayed profound paired pulse facilitation. Moreover, third and fourth spikes in a presynaptic burst also evoked large responses. This facilitation was associated with a decrease in the proportion of apparent failures in transmission and a change in the shape of the excitatory postsynaptic potential amplitude distribution, both indicative of an increase in the probability of transmitter release. However a large change in the mean amplitude was not associated with a similar change in the inverse square of the coefficient of variation. The result of this third test, taken in isolation, might therefore suggest that quantal amplitude had increased with paired-pulse facilitation. However, of the three tests applied, this last is the most heavily model-dependent and produced a result inconsistent with the results of the other two tests. The possibility is therefore discussed that both the shape of the excitatory postsynaptic potential amplitude distribution and the failure of coefficient of variation analysis to detect an apparently presynaptic change might result from the release at these synapses being poorly fit by a simple model. Based on a more complex model of synaptic release proposed by Faber and Korn [Faber and Korn (1991) Biophys. J. 60, 1288-1294] and a hypothesis proposed by Scharfman et al. [Scharfman et al. (1990) Neuroscience 37, 693-707], two hypotheses arising from the present study are discussed: (i) that branch point failure contributes to the pattern of synaptic activation at these connections; and (ii) that both presynaptic pyramidal firing pattern and axonal geometry contribute to the selection of the type of postsynaptic neurone preferentially activated.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationships between morphology and physiology of pyramid-pyramid single axon connections in rat neocortex in vitro.

1. Double intracellular recordings were made from 1163 pairs of pyramidal neurones in layer V‐VI of the rat somatomotor cortex in vitro using sharp electrodes filled with biocytin. Monosynaptically connected pairs of cells were identified when an action potential in one could elicit a constant latency excitatory postsynaptic potential (EPSP) in the other and the cells were filled with biocytin. Labelled cells were subsequently identified histologically with avidin‐horseradish peroxidase. 2. Thirty‐four pairs of cells were found to be monosynaptically connected. Fifteen of these pairs were sufficiently stable for electrophysiological recordings and three of these were recovered sufficiently to permit full morphological reconstruction. 3. The EPSP recorded between the first pair of pyramids varied in amplitude between 0 and 3 mV (mean 1.33 +/‐ 1.06 mV) and fluctuated considerably (coefficient of variation, 0.796). This was largely due to a high incidence of apparent failures of transmission. On reconstruction two boutons from the presynaptic pyramid axon were in close apposition to the proximal portions of basal dendrites of the postsynaptic cell. 4. In the second pair of pyramids the EPSP had a mean amplitude of 1.06 mV, and displayed a 10‐90% rise time of 2.8 ms and a width at half‐amplitude of 23 ms. This EPSP did not alter significantly with changes in membrane potential at the soma. The presynaptic axon closely apposed the distal apical dendrite of the postsynaptic cell in eight places. 5. In the third pair of pyramids, the EPSPs, recorded at a relatively depolarized membrane potential, were long lasting and could elicit slow dendritic spikes with long and variable latencies. These slow spikes suggested that the postsynaptic recording site was dendritic and on reconstruction a possible location was identified on the apical dendrite. A total of five presynaptic boutons closely apposed three separate, proximal branches of the postsynaptic apical dendrite. 6. These results provide the first illustration of a morphological basis for variations in functional properties of pyramid‐pyramid connections in the neocortex.

CA1 pyramidal to basket and bistratified cell EPSPs: dual intracellular recordings in rat hippocampal slices.

1 Dual intracellular recordings in the CA1 region of adult rat hippocampal slices and biocytin filling of synaptically connected cells were used to study the excitatory postsynaptic potentials (EPSPs) elicited in basket (n= 7) and bistratified interneurones (n= 7) by action potentials activated in simultaneously recorded pyramidal cells. 2 Interneurones could be subdivided according to their electrophysiological properties into classical fast spiking, burst firing, regular spiking and fast spiking cells with a rounded spike after‐hyperpolarization. These physiological classes did not, however, correlate with morphological type. EPSPs were not recorded in regular spiking cells. 3 Average EPSP amplitudes were larger in bistratified cells (range, 0.5–9 mV) than in basket cells (range, 0.15–3.6 mV) and the probability of obtaining a pyramidal cell‐interneurone EPSP was also higher for the bistratified cells (1:7) than for the basket cells (1:22). EPSP 10–90% rise times in bistratified cells (0.7–2 ms) and their widths at half‐amplitude (3.9–11.2 ms) were slightly longer than in basket cells (rise times, 0.4–1.6 ms; half‐widths, 2.2–9.7 ms). 4 The majority of these EPSPs (6 of 8 tested) increased in amplitude and duration with postsynaptic depolarization, although in two (of 4) basket cells the voltage relation was conventional. 5 All EPSPs tested in both basket (n= 7) and bistratified cells (n= 5) decreased in amplitude with repetitive presynaptic firing. The average amplitudes of second EPSPs elicited within 15 ms of the first were between 34 and 94% of the average amplitude of the first EPSP. Third and fourth EPSPs in brief trains were further depressed. This depression was associated with an increase in the incidence of apparent failures of transmission indicating a presynaptic locus.

Role of Olivary Electrical Coupling in Cerebellar Motor Learning

The level of electrotonic coupling in the inferior olive is extremely high, but its functional role in cerebellar motor control remains elusive. Here, we subjected mice that lack olivary coupling to paradigms that require learning-dependent timing. Cx36-deficient mice showed impaired timing of both locomotion and eye-blink responses that were conditioned to a tone. The latencies of their olivary spike activities in response to the unconditioned stimulus were significantly more variable than those in wild-types. Whole-cell recordings of olivary neurons in vivo showed that these differences in spike timing result at least in part from altered interactions with their subthreshold oscillations. These results, combined with analyses of olivary activities in computer simulations at both the cellular and systems level, suggest that electrotonic coupling among olivary neurons by gap junctions is essential for proper timing of their action potentials and thereby for learning-dependent timing in cerebellar motor control.