In vitro synaptogenesis between the somata of identified Lymnaea neurons requires protein synthesis but not extrinsic growth factors or substrate adhesion molecules

Nerve growth factors, substrate and cell adhesion molecules, and protein synthesis are considered necessary for most developmental programs, including cell proliferation, migration, differentiation, axogenesis, pathfinding, and synaptic plasticity. Their direct involvement in synapse formation, however, has not yet been fully determined. The neurite outgrowth that precedes synaptogenesis is contingent on protein synthesis, the availability of externally supplied growth factors, and substrate adhesion molecules. It is therefore difficult to ascertain whether these factors are also needed for synapse formation. To examine this issue directly we reconstructed synapses between the cell somata of identified Lymnaea neurons. We show that when paired in the presence of brain conditioned medium (CM), mutual inhibitory chemical synapses between neurons right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD4) formed in a soma-soma configuration (86%; n = 50). These synapses were reliable and target cell specific and were similar to those seen in the intact brain. To test whether synapse formation between RPeD1 and VD4 required de novo protein synthesis, the cells were paired in the presence of anisomycin (a nonspecific protein synthesis blocker). Chronic anisomycin treatment (18 hr) after cell pairing completely blocked synaptogenesis between RPeD1 and VD4 (n = 24); however, it did not affect neuronal excitability or responsiveness to exogenously applied transmitters (n = 7), nor did chronic anisomycin treatment affect synaptic transmission between pairs of cells that had formed synapses (n = 5). To test the growth and substrate dependence of synapse formation, RPeD1 and VD4 were paired in the absence of CM [defined medium; (n = 22)] on either plain plastic culture dishes (n = 10) or glass coverslips (n = 10). Neither CM nor any exogenous substrate was required for synapse formation. In summary, our data provide direct evidence that synaptogenesis in this system requires specific, cell contact-induced, de novo protein synthesis but does not depend on extrinsic growth factors or substrate adhesion molecules.     

High-performance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass

Individual GABAergic interneurons in hippocampus can powerfully inhibit more than a thousand excitatory pyramidal neurons. Therefore, control of interneuron excitability provides control over hippocampal networks. We have identified a novel mechanism in hippocampus that weakens excitatory synapses onto GABAergic interneurons. Following stimulation that elicits long-term potentiation at neighboring synapses onto excitatory cells, excitatory synapses onto inhibitory interneurons undergo a long-term synaptic depression (interneuron LTD; iLTD). Unlike most other forms of hippocampal synaptic plasticity, iLTD is not synapse specific: stimulation of an afferent pathway triggers depression not only of activated synapses but also of inactive excitatory synapses onto the same interneuron. These results suggest that high frequency afferent activity increases hippocampal excitability through a dual mechanism, simultaneously potentiating synapses onto excitatory neurons and depressing synapses onto inhibitory neurons.     

Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons

Cell cultures were used to analyze the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the development of synaptic transmission. Neurons obtained from embryonic day 18 (E18) rat hippocampus and cultured for 2 weeks exhibited extensive spontaneous synaptic activity. By comparison, neurons obtained from E16 hippocampus expressed very low levels of spontaneous or evoked synaptic activity. Neurotrophin treatment produced a sevenfold increase in the number of functional synaptic connections in the E16 cultures. BDNF induced formation of both excitatory and inhibitory synapses, whereas NT-3 induced formation of only excitatory synapses. These effects were independent of serum or the age of the glia bed used for the culture. They were not accompanied by significant changes in synaptic-vesicle-associated proteins or glutamate receptors. Treatment of the cultures with the neurotrophins for 3 d was sufficient to establish the maximal level of functional synapses. During this period, neurotrophins did not affect the viability or the morphology of the excitatory neurons, although they did produce an increase in the number and length of dendrites of the GABAergic neurons. Remarkably, only BDNF caused an increase in the number of axonal branches and in the total length of the axons of the GABAergic neurons. These results support a unique and differential role for neurotrophins in the formation of excitatory and inhibitory synapses in the developing hippocampus.     

Cat intraamygdaloid inhibitory network: ultrastructural organization of parvalbumin-immunoreactive elements

Projection neurons of the basolateral (BL) amygdaloid complex are regulated by an intrinsic inhibitory network. To improve our understanding of this inhibitory circuit, we studied the synaptology of parvalbumin-immunopositive (PV+) elements as this calcium-binding protein is localized in a subpopulation of gamma-aminobutyric acid (GABA)-ergic interneurons. Two populations of PV+ cells were identified on the basis of soma shape (ovoid, type A vs. polygonal, type B). In the lateral and BL nuclei, the majority of boutons in contact with PV+ cells formed asymmetric synapses (types 1-3; 94%), whereas a minority (type 4, 6%) established symmetric synaptic contacts and resembled GABAergic terminals. In both nuclei, type B PV+ perikarya were more densely innervated than were type A neurons. However, the pattern of synaptic innervation of type B PV+ neurons differed in the two nuclei: in the lateral nucleus, they were almost exclusively innervated by a population of small, presumed excitatory terminals (type 1), whereas the four categories of terminals contributed more equally to their innervation in the BL nucleus. PV+ boutons belonged to a single category of terminals that was enriched with GABA and formed symmetric synapses mostly with the proximal part of PV neurons. The proportion of axosomatic synapses was significantly higher in the lateral nucleus than in the BL nucleus (33% vs. 18%). The reverse was true for the contacts with proximal dendrites (33% in the lateral nucleus vs. 46% in the BL nucleus). The remaining terminals formed synapses with distal dendrites (23-28%) and spines (8-12%). These results indicate that PV+ interneurons receive massive excitatory inputs and that PV+ terminals are strategically located to exert a powerful inhibitory control of amygdala neurons.     

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3 x 10(-7) M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10(-10) to 10(-9) M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca2+-free and high-Mg2+ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V1-receptor agonist and completely blocked by a selective V1-antagonist. V2-agonist produced no effects on ACe neurons and V2-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V1-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.     

Cerebral serotonergic neurons reciprocally modulate swim and withdrawal neural networks in the mollusk Clione limacina

1. A pair of serotonin-immunoreactive neurons has been identified in the cerebral ganglia of the pteropod mollusk Clione limacina, which produce coordinated, excitatory/inhibitory effects on neurons controlling two incompatible behaviors, swimming and whole body withdrawal. These cells were designated cerebral serotonergic ventral (Cr-SV) neurons. 2. Activation of Cr-SV neurons produces a prominent inhibition of the pleural withdrawal neurons, which have been previously shown to induce whole body withdrawal in Clione. In addition, the cerebral neurons produce weak excitatory inputs to swim motor neurons, pedal serotonergic neurons involved in the peripheral modulation of swimming, and to the serotonergic heart excitor neuron. 3. Inhibitory and excitatory effects appear to be produced by serotonin because they are mimicked by exogenous serotonin and are blocked by the serotonin antagonist mianserin. 4. All serotonergic neurons identified thus far in the CNS of Clione appear to function in a coordinated manner, altering a variety of neural centers all directed toward the activation of swimming behavior.     

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala. Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium. Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABA(A)-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning.     

N-cadherin redistribution during synaptogenesis in hippocampal neurons

Cadherins are homophilic adhesion molecules that, together with their intracellular binding partners the catenins, mediate adhesion and signaling at a variety of intercellular junctions. This study shows that neural (N)-cadherin and beta-catenin, an intracellular binding partner for the classic cadherins, are present in axons and dendrites before synapse formation and then cluster at developing synapses between hippocampal neurons. N-cadherin is expressed initially at all synaptic sites but rapidly becomes restricted to a subpopulation of excitatory synaptic sites. Sites of GABAergic, inhibitory synapses in mature cultures therefore lack N-cadherin but are associated with clusters of beta-catenin, implying that they contain a different classic cadherin. These findings indicate that N-cadherin adhesion may stabilize early synapses that can then be remodeled to express a different cadherin and that cadherins systematically differentiate between functionally (excitatory and inhibitory) and spatially distinct synaptic sites on single neurons. These results suggest that differential cadherin expression may orchestrate the point-to-point specificity displayed by developing synapses.     

Trophic and contact conditions modulate synapse formation between identified neurons

We tested the ability of an identified interneuron from the mollusk, Lymnaea stagnalis, to reestablish appropriate synapses in vitro. In the CNS, the giant dopaminergic neuron, designated as right pedal dorsal one (RPeD1), makes an excitatory, chemical synapse with a pair of essentially identical postsynaptic cells known as visceral dorsal two and three (VD2/3). When the somata of the pre- and postsynaptic neurons were juxtaposed and cultured in vitro in defined medium, i.e. , a soma-soma synapse, only an inappropriate electrical synapse was observed. The postsynaptic cell still responded to applied dopamine, the presynaptic transmitter, indicating that the lack of chemical synapse formation was not due to lack of dopamine receptors. When the somata were cultured apart in conditioned medium (medium previously incubated with Lymnaea CNS, thereby deriving trophic factors), the cells exhibited overlapping neurite outgrowth that resulted in an appropriate excitatory, chemical synapse from RPeD1 to VD2/3. On the other hand, when the cell pair was cultured in a soma-soma configuration, but in conditioned medium, a mixed chemical-electrical synapse was observed. Because conditioned medium could partially overcome the limitations of the soma-soma configuration and initiate chemical synapse formation, this data suggests that conditioned medium contains a factor(s) that supports synaptogenesis.     

Transient and sustained expression of FMRFamide-like immunoreactivity in the developing nervous system of Lymnaea stagnalis (Mollusca, Pulmonata)

1. In the present study we have investigated the ontogeny of FMRFamide expression in the snail, Lymnaea stagnalis, from its first appearance to its distribution in young adults. 2. The first FMRFamide-like immunoreactive (FaLI) cells within CNS appear by E45 embryonic stage (premetamorphic veliger). The number of FaLI neurons increases throughout both pre- and post-hatching development. 3. Both transient and sustained expression of FMRFamide-like immunoreactivity by specific sets of neurons occurs. Two cells which transiently express immunoreactivity appear outside the future CNS by the stage E45. Other population of transient FaLI neurons includes bilaterally symmetric groups of cells in the cerebral and pedal ganglia during posthatching stages P1 (hatchlings) to P5 (juveniles). All other immunostained cells which appear during development maintain their transmitter phenotype into adulthood. 4. The possible role of FMRFamide-related peptides in the processes of morpho- and neurogenesis is discussed.     

Differential glutamatergic innervation in cytochrome oxidase-rich and -poor regions of the macaque striate cortex: quantitative EM analysis of neurons and neuropil

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase (CO)-rich puffs and CO-poor interpuffs in its supragranular layers. However, the neurochemical basis for their differences in metabolic activity and physiological properties is not well understood. The goals of the present study were to determine whether CO levels in postsynaptic neuronal compartments were correlated with the proportion of excitatory glutamate-immunoreactive (Glu-IR) synapses they received and if Glu-IR terminals and synapses in puffs differed from those in interpuffs. By combining CO histochemistry and postembedding Glu immunocytochemistry on the same ultrathin sections, the simultaneous distribution of the two markers in individual neuronal profiles was quantitatively analyzed. As a comparison, adjacent sections were identically processed for the double labeling of CO and GABA, an inhibitory neurotransmitter. In both puffs and interpuffs, most axon terminals forming asymmetric synapses (84%)--but not symmetric ones, which were GABA-IR--were intensely immunoreactive for Glu. GABA-IR neurons received mainly Glu-IR synapses on their cell bodies, and they had three times as many mitochondria darkly reactive for CO than Glu-rich neurons, which received only GABA-IR axosomatic synapses. In puffs, GABA-IR neurons received a significantly higher ratio of Glu-IR to GABA-IR axosomatic synapses and contained about twice as many darkly CO-reactive mitochondria than those in interpuffs. There were significantly more Glu-IR synapses and a higher ratio of Glu- to GABA-IR synapses in the neuropil of puffs than of interpuffs. Moreover, Glu-IR axon terminals in puffs contained approximately three times more darkly CO-reactive mitochondria than those in interpuffs, suggesting that the former may be synaptically more active. Thus, the present results are consistent with our hypothesis that the levels of oxidative metabolism in postsynaptic neurons and neuropil are positively correlated with the proportion of excitatory synapses they receive. Our findings also suggest that excitatory synaptic activity may be more prominent in puffs than in interpuffs, and that the neurochemical and synaptic differences may constitute one of the bases for physiological and functional diversities between the two regions.     

Loss of inhibitory synapses on the soma and axon initial segment of pyramidal cells in human epileptic peritumoural neocortex: implications for epilepsy

The peritumoural neocortex removed from epileptic patients represents an important region for research because of its possible relationship to the generation, maintenance, and propagation of seizures. The peritumoural neocortex removed from an epileptic patient showing a regrowth of an anaplastic astrocytoma was examined in detail using immunocytochemistry for gamma-aminobutyric acid, glutamic acid decarboxylase, parvalbumin, nonphosphorylated neurofilament protein, glial fibrillary acidic protein, and histocompatibility antigen HLA-DR. The patterns of immunostaining were compared with the cytoarchitecture and myeloarchitecture in adjacent sections, and with the patterns of immunostaining observed in normal control neocortex. Furthermore, quantitative electron microscopy was used to compare the synaptic densities of presumptive excitatory and inhibitory synapses between regions showing different grades of cytoarchitectural and neurochemical alterations in the peritumoural neocortex, and to compare these regions with normal neocortex. A variety of changes in synaptic circuits in the peritumoural neocortex was found, but it appears that neurons within the less abnormal-looking regions were involved in altered synaptic circuits that might contribute to epileptic activity. In these regions, the most prominent change was the loss of inhibitory synapses on the soma and axon initial segment of pyramidal cells, but numerous excitatory synapses were present on their dendrites that would make these neurons hyperexcitable. However, the most abnormal regions histologically were likely a primary zone for progression of the tumour, with many surviving neurones, but which received and formed very few synapses; thus, they were probably unrelated to the initiation, maintenance, or propagation of seizures.     

Monaural spectral contrast mechanism for neural sensitivity to sound direction in the medial geniculate body of the cat

Monaural spectral contrast mechanism for neural sensitivity to sound direction in the medial geniculate body of the cat. J. Neurophysiol. 78: 2754-2771, 1997. Central auditory neurons vary in sound direction sensitivity. Insensitive cells discharge well to all sound source directions, whereas sensitive cells discharge well to certain directions and poorly to others. High-frequency neurons in the latter group are differentially sensitive to binaural and monaural directional cues present in broadband noise (BBN). Binaural directional (BD) cells require binaural stimulation for directional sensitivity; monaural directional (MD) cells are sensitive to the direction of monaural stimuli. A model of MD sensitivity was tested using single-unit responses. The model assumes that MD cells derive directional sensitivity from pinna-derived spectral cues (head related transfer function, HRTF). This assumption was supported by the similarity of effects that pinna orientation produces on locations of HRTF patterns and on locations of MD cell azimuth function peaks and nulls. According to the model, MD neurons derive directional sensitivity by use of excitatory/inhibitory antagonism to compare sound pressure in excitatory and inhibitory frequency domains, and a variety of observations are consistent with this idea. 1) Frequency response areas of MD cells consist of excitatory and inhibitory domains. MD cells exhibited a higher proportion of multiple excitatory domains and narrower excitatory frequency domains than BD cells, features that may reflect specialization for spectral-dependent directional sensitivity. 2) MD sensitivity requires sound pressure in excitatory and inhibitory frequency domains. Directional sensitivity was evaluated using stimuli with frequency components confined exclusively to excitatory domains (E-only stimuli) or distributed in both excitatory and inhibitory domains (E/I stimuli). Each of 13 MD cells that were tested exhibited higher directional sensitivity to E/I than to E-only stimuli; most MD cells exhibited relatively low directional sensitivity when frequency components were confined exclusively to excitatory domains. 3) MD sensitivity derives from excitatory/inhibitory antagonism (spectral inhibition). Comparison of responses to best frequency and E/I stimuli provided strong support for spectral inhibition. Although spectral facilitation conceivably could contribute to directional sensitivity with direction-dependent increases in response, the results did not show this to be a significant factor. 4) Direction-dependent decreases in responsiveness to BBN reflect increased sound pressure in inhibitory relative to excitatory frequency domains. This idea was tested using the strength of two-tone inhibition, which is a function of stimulus levels in inhibitory relative to excitatory frequency domains. The finding that two-tone inhibition was stronger at directions where BBN responses were minimal than at directions where they were maximal supports the model.     

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.     

Dopaminergic transmission between identified neurons from the mollusk, Lymnaea stagnalis

1. Dopaminergic transmission was investigated in the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis. 2. The giant pedal neuron, designated as right pedal dorsal one (RPeD1), makes chemical, monosynaptic connections with a number of identified follower cells in the CNS. Previous work has shown that RPeD1 is an interneuron and a important component of the Lymnaea respiratory central pattern generator. In this study, the hypothesis that RPeD1 uses dopamine as its neurotransmitter was tested by chromatographic, pharmacological, and electrophysiological methods. Characterization of RPeD1's transmitter pharmacology is essential to clearly understand its role in Lymnaea. 3. Earlier studies demonstrated that the soma of RPeD1 contains dopamine. This was quantitated in the present study by high-performance liquid chromatography (with electrochemical detection) of isolated RPeD1 somata and growth cones, which yielded 0.8 +/- 0.3 and 0.10 +/- 0.08 pmol of dopamine per soma and growth cone, respectively. 4. Bath or pressure application of dopamine to follower cells of RPeD1, in situ, mimicked the effects of RPeD1 stimulation. Dose-response curves were constructed for the excitatory effect of dopamine on follower cells, visceral dorsal two and three (VD2/3) (ED50 = 39 microM; Hill coefficient = 1.03), and the inhibitory effect of dopamine on follower cell, visceral dorsal four (ED50 = 33 microM; Hill coefficient = 0.92). 5. The following dopamine agonists (100 microM) were tested by bath application: 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), apopmorphine, 2-bromo-alpha-ergocryptine, deoxyepinephrine (DE), mesulergine, (-) quinpirole, SKF 38393, and tyramine. Only the general dopamine agonists, ADTN and DE, mimicked RPeD1's effects on its follower cells. 6. When VD2/3 was isolated and plated in vitro, it maintained a depolarizing response to dopamine. This response was reduced by intracellular injection of the G-protein blocker, GDP-beta-S (2 mM in electrode). Similarly, incubation of VD2/3, in vitro for approximately 18 h, with pertussis toxin (PTX; 5 micrograms/ml), the G-protein inactivating exotoxin, also reduced the dopamine response. Injecting GDP or incubating in heat-inactivated PTX did not effect the response. 7. Several dopamine antagonists were used in an attempt to block RPeD1's synapses: chlorpromazine, ergonovine, fluphenazine, haloperidol, 6-hydroxydopamine, SCH 23390, (+/-) sulpiride, and tubocurarine. Only the D-2 dopamine receptor antagonist, (+/-) sulpiride, reversibly blocked synaptic transmission from RPeD1 to its follower cells. Both the (+) and the (-) enantiomer of sulpiride also antagonized synaptic transmission. A dose-inhibition curve for (+/-) sulpiride was constructed (IC50 = 47 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Extrastriate feedback to primary visual cortex in primates: a quantitative analysis of connectivity

Knowledge-based or top-down influences on primary visual cortex (area V1) are believed to originate from information conveyed by extrastriate feedback axon connections. Understanding how this information is communicated to area V1 neurons relies in part on elucidating the quantitative as well as the qualitative nature of extrastriate pathway connectivity. A quantitative analysis of the connectivity based on anatomical data regarding the feedback pathway from extrastriate area V2 to area V1 in macaque monkey suggests (i) a total of around ten million or more area V2 axons project to area V1; (ii) the mean number of synaptic inputs from area V2 per upper-layer pyramidal cell in area V1 is less than 6% of all excitatory inputs; and (iii) the mean degree of convergence of area V2 afferents may be high, perhaps more than 100 afferent axons per cell. These results are consistent with empirical observations of the density of radial myelinated axons present in the upper layers in macaque area V1 and the proportion of excitatory extrastriate feedback synaptic inputs onto upper-layer neurons in rat visual cortex. Thus, in primate area V1, extrastriate feedback synapses onto upper-layer cells may, like geniculocortical afferent synapses onto layer IVC neurons, form only a small percentage of the total excitatory synaptic input.     

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.     

Inhibition synchronizes sparsely connected cortical neurons within and between columns in realistic network models

Networks of compartmental model neurons were used to investigate the biophysical basis of the synchronization observed between sparsely-connected neurons in neocortex. A model of a single column in layer 5 consisted of 100 model neurons: 80 pyramidal and 20 inhibitory. The pyramidal cells had conductances that caused intrinsic repetitive bursting at different frequencies when driven with the same input. When connected randomly with a connection density of 10%, a single model column displayed synchronous oscillatory action potentials in response to stationary, uncorrelated Poisson spike-train inputs. Synchrony required a high ratio of inhibitory to excitatory synaptic strength; the optimal ratio was 4 : 1, within the range observed in cortex. The synchrony was insensitive to variation in amplitudes of postsynaptic potentials and synaptic delay times, even when the mean synaptic delay times were varied over the range 1 to 7 ms. Synchrony was found to be sensitive to the strength of reciprocal inhibition between the inhibitory neurons in one column: Too weak or too strong reciprocal inhibition degraded intra-columnar synchrony. The only parameter that affected the oscillation frequency of the network was the strength of the external driving input which could shift the frequency between 35 to 60 Hz. The same results were obtained using a model column of 1000 neurons with a connection density of 5%, except that the oscillation became more regular. Synchronization between cortical columns was studied in a model consisting of two columns with 100 model neurons each. When connections were made with a density of 3% between the pyramidal cells of each column there was no inter-columnar synchrony and in some cases the columns oscillated 180 degrees out of phase with each other. Only when connections from the pyramidal cells in each column to the inhibitory cells in the other column were added was synchrony between the columns observed. This synchrony was established within one or two cycles of the oscillation and there was on average less than 1 ms phase difference between the two columns. Unlike the intra-columnar synchronization, the inter-columnar synchronization was found to be sensitive to the synaptic delay: A mean delay of greater than 5 ms virtually abolished synchronization between columns.     

In vivo activity-dependent plasticity at cortico-striatal connections: evidence for physiological long-term potentiation

The purpose of the present study was to investigate in vivo the activity-dependent plasticity of glutamatergic cortico-striatal synapses. Electrical stimuli were applied in the facial motor cortex and intracellular recordings were performed in the ipsilateral striatal projection field of this cortical area. Recorded cells exhibited the typical intrinsic membrane properties of striatal output neurons and were identified morphologically as medium spiny type I neurons. Subthreshold cortical tetanization produced either short-term posttetanic potentiation or short-term depression of cortically-evoked excitatory postsynaptic potentials. When coupled with a postsynaptic depolarization leading the membrane potential to a suprathreshold level, the tetanus induced long-term potentiation (LTP) of cortico-striatal synaptic transmission. Induction of striatal LTP was prevented by intracellular injection of a calcium chelator suggesting that this synaptic plasticity involves an increase of postsynaptic free calcium concentration. Contrasting with previous in vitro studies our findings demonstrate that LTP constitutes the normal form of use-dependent plasticity at cortico-striatal synapses. Since excitation of striatal neurons produces a disinhibition of premotor networks, LTP at excitatory striatal inputs should favor the initiation of movements and therefore could be critical for the functions of basal ganglia in motor learning.     

Synapses from labeled type II axons in the mouse cochlear nucleus

This study investigates the ultrastructure and central targets in the cochlear nucleus of axonal swellings of type II primary afferent neurons. Type II axons comprise only 5-10% of the axons of the auditory nerve of mammals, but they alone provide the afferent innervation of the outer hair cells. In this study, type II axons were labeled with horseradish peroxidase, and serial-section electron microscopy was used to examine their swellings in: (1) the granule-cell lamina at its boundary with posteroventral cochlear nucleus, (2) the rostral anteroventral cochlear nucleus, and (3) the auditory nerve root. Only some (18%) of the type II terminal and en-passant swellings formed synapses. The synapses were asymmetric and contained clear round synaptic vesicles, suggesting that they are excitatory. Type II synapses were compared to those from type I fibers providing the afferent innervation of the inner hair cells. Type II synapses tended to have slightly smaller and fewer synaptic vesicles, had a greater proportion of the membrane apposition accompanied by a postsynaptic density, and often had densities that were discontinuous or 'perforated'. In all cochlear nucleus regions examined, the postsynaptic targets of type II synapses had characteristics of dendrites; in most cases these dendrites could not be traced to their cell bodies of origin. Some evidence suggests, however, that targets may include granule cells, spherical cells, and other cells in the nerve root. These results suggest afferent information from outer hair cells reaches diverse regions and targets within the cochlear nucleus.