Synaptogenesis by single identified neurons in vitro: contribution of rapidly transported and newly synthesized proteins

The object of these studies is to define the molecular events that occur during synaptogenesis. Our approach is to use single identified Aplysia neurons grown in culture under conditions where chemical synapses are formed. In this report we studied synapses established by R2, a giant cholinergic neuron, onto neurons R15 and L11, and a group of left upper quadrant (LUQ) cells. The detailed electrophysiology of these contacts was described in the preceding paper (Schacher, S., S. G. Rayport, and R. T. Ambron (1985) (J. Neurosci. 5: 2851-2856). Within the animal, R2 synapses on thousands of unicellular mucus glands in the skin. R2 growing in vitro will establish contacts with isolated mucus glands. Although we do not know whether a functional synapse is formed, electron microscopy shows that the membrane in the area of contact is differentiated and that the ending is filled with various types of vesicles. A single R2 regenerating neurites in vitro synthesizes more than 300 polypeptides containing [35S]methionine. Many of these are subsequently transported into the growing neurites. We compared the newly synthesized proteins made by R2 before and after synapse formation and found that the expression of a 68-kilodalton (kd) and 72-kd protein was markedly enhanced after synaptogenesis. The finding that only two proteins were affected implies that many of the proteins required for synapse formation are present in R2 prior to contacting a target cell. Support for this idea was obtained when we compared the proteins present in R2's neurites in vitro with those that are rapidly transported to R2's mature synapses in vivo (Ambron, R. T., S. Schacher, and S. G. Rayport (1985) J. Neurosci. 5: 2866-2873).(ABSTRACT TRUNCATED AT 250 WORDS)     

Innervation of the kidney of Aplysia by L10, the LUQ cells, and an identified peripheral motoneuron

The purpose of this study was to begin to describe the neural circuit within the abdominal ganglion that modulates renal functioning in Aplysia. We found that the previously described cholinergic neuron L10 and peptidergic left upper quadrant (LUQ) neurons have important roles in the control of the kidney. Cell L10 and a subset of the LUQ cells branch extensively within the kidney and send major processes to the renal pore, a sphincter that controls the efflux of urine. The renal pore has circular (closer) and radial (opener) muscle fibers that act as antagonists. Embedded within the wall of the renal pore is a newly identified peripheral neuron, RPO, which is a renal pore opener motoneuron. L10 activity causes opening of the renal pore by directly exciting pore opener muscle, inhibiting closer muscle, and exciting RPO. When RPO is active, it generates synchronous, discrete twitches in the opener muscle fibers. The action potentials recorded in RPO exhibit pronounced broadening at physiological rates of firing. LUQ cells that project to the renal pore cause it to close, and they antagonize the opening generated by an L10 burst. The pore closing caused by the LUQ cells is mediated in part by heterosynaptic inhibition of the L10 to RPO excitatory connection. The previously described central inhibitory connections from L10 to the LUQ cells ensure that these 2 classes of antagonists fire out of phase with each other. Our data, along with those from earlier studies demonstrating that L10 plays an important role in controlling the circulatory system, suggest that L10 and the LUQ cells modulate various aspects of renal function in Aplysia, including filtration and micturition.     

Identified Aplysia neurons form specific chemical synapses in culture

Identified neurons from the abdominal ganglion of the marine mollusc Aplysia californica make specific transmitter-mediated synapses in dissociated cell culture. The cholinergic interneuron L10 makes synapses in vitro with one group of its follower cells, the left upper quadrant cells, and these connections exhibit the features of these synapses in vivo when the postsynaptic cells are plated with their initial axon segments. Furthermore, L10 will avoid making synapses with right upper quadrant cells, which contain cholinergic receptors but do not synapse with L10 in vivo. This in vitro system can therefore be used as a model in which to study the development of specific neuronal connections.     

A vasoactive intestinal peptide-like cotransmitter at cholinergic synapses between rat myenteric neurons in cell culture

Intracellular recording and immunochemical techniques were used to study synaptic transmission between individual pairs of rat myenteric plexus neurons in cell culture. This report describes the synaptic connections made by "dual function" presynaptic neurons that evoked slow postsynaptic depolarizations (slow EPSPs) in the same neurons in which they also evoked fast nicotinic cholinergic EPSPs. The slow EPSPs occurred only when presynaptic neurons were stimulated at frequencies of 5 Hz or higher. During the slow EPSPs, slope input resistance increased. The slow EPSPs were not detectably voltage-dependent, and they reversed sign at the estimated K+ equilibrium potential, suggesting that they resulted from a synaptically mediated decrease in resting K+ conductance. Several lines of evidence suggested that dual-function neurons evoke slow EPSPs by releasing a vasoactive intestinal peptide (VIP)-like cotransmitter. (1) Immunocytochemical staining revealed VIP-like immunoreactivity in all physiologically identified dual-function neurons. (2) Responses to exogenously applied VIP mimicked the slow EPSPs. (3) Superfusion of cultures with anti-VIP antisera blocked the slow EPSPs reversibly, as did application of desensitizing doses of VIP. These findings suggest that during periods of increased activity, subsets of cholinergic myenteric neurons release a VIP-like cotransmitter that enhances postsynaptic excitability. The effects of the cotransmitter may help to compensate for decreases in nicotinic EPSPs that occur during increased presynaptic activity.     

Novel fast adapting interneurons mediate cholinergic‐induced fast GABAA inhibitory postsynaptic currents in striatal spiny neurons

Previous work suggests that neostriatal cholinergic interneurons control the activity of several classes of GABAergic interneurons through fast nicotinic receptor‐mediated synaptic inputs. Although indirect evidence has suggested the existence of several classes of interneurons controlled by this mechanism, only one such cell type, the neuropeptide‐Y‐expressing neurogliaform neuron, has been identified to date. Here we tested the hypothesis that in addition to the neurogliaform neurons that elicit slow GABAergic inhibitory responses, another interneuron type exists in the striatum that receives strong nicotinic cholinergic input and elicits conventional fast GABAergic synaptic responses in projection neurons. We obtained in vitro slice recordings from double transgenic mice in which Channelrhodopsin‐2 was natively expressed in cholinergic neurons and a population of serotonin receptor‐3a‐Cre‐expressing GABAergic interneurons were visualized with tdTomato. We show that among the targeted GABAergic interneurons a novel type of interneuron, termed the fast‐adapting interneuron, can be identified that is distinct from previously known interneurons based on immunocytochemical and electrophysiological criteria. We show using optogenetic activation of cholinergic inputs that fast‐adapting interneurons receive a powerful supra‐threshold nicotinic cholinergic input in vitro. Moreover, fast adapting neurons are densely connected to projection neurons and elicit fast, GABA_A receptor‐mediated inhibitory postsynaptic current responses. The nicotinic receptor‐mediated activation of fast‐adapting interneurons may constitute an important mechanism through which cholinergic interneurons control the activity of projection neurons and perhaps the plasticity of their synaptic inputs when animals encounter reinforcing or otherwise salient stimuli.     

Synaptic connections in the buccal ganglia of Aplysia mediated by an identified neuron containing a CCK/gastrin-like peptide co-localized with acetylcholine

The identified neuron, B13, located bilaterally in the buccal ganglion of the marine mollusc Aplysia californica, contains a classical neurotransmitter (acetylcholine) and a cholecystokinin/gastrin-like (CCK/G-li) peptide. The following study demonstrates that B13 makes direct synaptic connections with several identifiable postsynaptic follower neurons. These follower neurons also receive convergent input from previously identified cholinergic neurons, B4 and B5, which do not contain a CCK/G-li peptide. The cholinergic responses mediated by B4/B5 and B13 are similar, including in at least one buccal follower, a two-component inhibitory response not seen in previous studies of the buccal ganglia circuits. However, when the cholinergic responses are blocked by appropriate antagonists, a residual, slow depolarizing, chemically-mediated response is observed in two of the identifiable followers when action potentials are evoked in B13 but not when action potentials are evoked in B4 or B5.     

Spontaneous activity of neostriatal cholinergic interneurons in vitro.

Neostriatal cholinergic interneurons fire irregularly but tonically in vivo. The summation of relatively few depolarizing potentials and their temporal sequence are thought to underlie spike triggering and the irregularity of action potential timing, respectively. In these experiments we used whole-cell, cell-attached, and extracellular recording techniques to investigate the role of spontaneous synaptic inputs in the generation and patterning of action potentials in cholinergic interneurons in vitro. Cholinergic cells were spontaneously active in vitro at 25 +/- 1 degrees C during whole-cell recording from 2 to 3 week postnatal slices and at 35 +/- 2 degrees C during cell-attached and extracellular recording from 3 to 4 week postnatal slices. A range of firing frequencies and patterns was observed including regular, irregular, and burst firing. Blockade of AMPA and NMDA receptors altered neither the firing rate nor the pattern, and accordingly, voltage-clamp data revealed a very low incidence of spontaneous EPSCs. GABAA receptor antagonists were also ineffective in altering the spiking frequency or pattern owing to minimal inhibitory input in vitro. Functional excitatory and inhibitory inputs to cholinergic cells were disclosed after application of 4-aminopyridine (100 microM), indicating that these synapses are present but not active in vitro. Blockade of D1 or D2 dopamine receptors or muscarinic receptors also failed to influence tonic activity in cholinergic cells. Together these data indicate that cholinergic interneurons are endogenously active and generate action potentials in the absence of any synaptic input. Interspike interval histograms and autocorrelograms generated from unit recordings of cholinergic cells in vitro were indistinguishable from those of tonically active neurons recorded in vivo. Irregular spiking is therefore embedded in the mechanism responsible for endogenous activity.     

Actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum.

We used intracellular electrophysiological recording to study the actions of noradrenaline on myenteric neurons in the guinea pig gastric antrum. Noradrenaline caused a dose-dependent inhibition of the stimulus-evoked cholinergic fast excitatory postsynaptic potentials (EPSPs). Noradrenaline had no effect on the postsynaptic response to acetylcholine, suggesting a presynaptic site of action. The slow EPSP was also presynaptically inhibited by noradrenaline. In only 5% of the neurons, noradrenaline caused a postsynaptic depolarization, accompanied by increased input resistance and enhanced excitability. Studies with adrenergic antagonists and agonists revealed that the presynaptic inhibitory effect was mediated by an alpha 2-receptor, while the postsynaptic excitatory effect seemed to be mediated by an alpha 1 receptor. We conclude that noradrenaline inhibits neurotransmitter release from cholinergic and non-cholinergic nerve terminals in the myenteric plexus of the antrum and that it excites a subpopulation of antral neurons. Both mechanisms may contribute to the neurally mediated inhibitory action of noradrenaline on gastric contractility.     

Tyrosine hydroxylase activity decreases with induction of cholinergic properties in cultured sympathetic neurons

Establishment of transmitter phenotype is an essential step in neuronal development. Studies on rat sympathetic neurons both in vivo and in vitro have provided evidence that mature cholinergic sympathetic neurons arise from previously noradrenergic neurons. Cultured rat superior cervical ganglion neurons can be influenced by their environment to remain noradrenergic, to acquire dual transmitter function, or to become predominantly cholinergic. Several other neuronal traits, such as a variety of surface molecules and released proteins, change simultaneously with levels of catecholamine and acetylcholine production, suggesting that various components of transmitter phenotype are regulated in concert. In this report, tyrosine hydroxylase levels are compared in neurons cultured under noradrenergic, dual function, or cholinergic conditions. Both enzyme activity in cell extracts and immunocytochemical staining were measured. These methods showed significantly less tyrosine hydroxylase enzyme activity and immunoreactive material in cholinergic cultures compared to noradrenergic and dual function cultures. These data support the interpretation that a switch in transmitter status from noradrenergic to cholinergic has occurred. This interpretation contrasts with that of Iacovitti et al. (Iacovitti, L., T. H. Joh, D. H. Park, and R. P. Bunge (1981) J. Neurosci. 1: 685-690), who conducted their experiments under critically different culture conditions.     

Comparison of central versus peripheral nerve pathways to the guinea pig inferior mesenteric ganglion determined electrophysiologically after chronic nerve section

The contributions of central versus peripheral nerve pathways to neurons of the inferior mesenteric ganglion of guinea pigs were studied. Nerve trunks innervating neurons in the ganglion were surgically sectioned and intracellular electrical responses to nerve stimulation were measured 6-8 days after surgery. Guinea pigs were divided into two experimental groups: (1) those that had the lumbar sympathetic chain ganglia (LSG) L2 through L4 removed and (2) those that had the intermesenteric, lumbar colonic and hypogastric nerves sectioned leaving central connections intact. After 6-8 days fast excitatory postsynaptic potentials (EPSPs) and slow EPSPs were recorded intracellularly in randomly selected principal ganglionic neurons. The threshold stimulus voltage to elicit a fast EPSP, the amplitude of the slow EPSP and the number of neurons in which each type of synaptic potential occurred in response to stimulation of each of the nerve trunks was compared between surgically-sectioned animals and sham-operated controls. Neither section of preganglionic nerve trunks nor of postganglionic nerve trunks eliminated all synaptic input to neurons in the ganglion, indicating that neurons with cell bodies located central to the ganglion as well as in visceral target organs made synaptic connections in the ganglion. Both fast and slow synaptic potentials could be evoked by stimulation of postganglionic nerve trunks even after they were sectioned provided that preganglionic nerves were intact, indicating that axons of central origin which synapse in the ganglion may continue out into postganglionic nerve trunks. In like manner, evidence was obtained indicating that fibers from peripheral nerve trunks which initiate either fast or slow synaptic potentials in ganglionic neurons may continue out into the lumbar splanchnic nerves. These studies demonstrate that the synaptic potentials recorded in the inferior mesenteric ganglion arise not only from neurons with cell bodies central to the ganglion but also from neurons with cell bodies located in the visceral organs which this ganglion subserves.     

Developmental changes in serotonin actions in rat hippocampus

Intracellular activity was recorded from neurons in immature rat hippocampal slices. The presence of intrinsic inhibitory synaptic potentials as well as responses to serotonin were assessed in slices of 1, 2 or 3 postnatal weeks of age. Young (1 week) cells had only a marginal hyperpolarizing response to serotonin and no detectable intrinsic inhibitory synaptic potentials. At 2 weeks of age neurons already expressed a fast IPSP (inhibitory postsynaptic potential). The responses to serotonin were different from those of adult cells in that they involved primarily a large decrease in input resistance with only small potential changes. In cells of this age serotonin caused a marked increase in spontaneous IPSP discharges and a blockade of a slow afterhyperpolarization. In 3-week-old rats the fast and slow components of the IPSP were present as in adult and the responses to serotonin included a large hyperpolarization associated with an increase in K conductance, a blockade of slow afterhyperpolarization and a blockade of a slow IPSP, as seen in adult cells. These results indicate that the complex pattern of reactivity to serotonin is differentially regulated in the developing brain.     

Ultrastructural localization of choline acetyltransferase in the rat rostral ventrolateral medulla: evidence for major synaptic relations with non-catecholaminergic neurons

Pharmacological and biochemical studies suggest that interactions between cholinergic and catecholaminergic neurons, particularly those of the C1 adrenergic cell group, in the rostral ventrolateral medulla (RVL) may be important in cardiovascular control. Ultrastructural localization of choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and its relation to neurons exhibiting immunoreactivity for catecholamine- (tyrosine hydroxylase; TH) or adrenaline (phenylethanolamine-N-methyltransferase; PNMT) -synthesizing enzymes were examined in the RVL using dual immunoautoradiographic and peroxidase anti-peroxidase (PAP) labeling methods. By light microscopy, the ChAT-immunoreactive neurons were located both dorsally (i.e. the nucleus ambiguus) and ventromedially to those labeled with TH or PNMT (TH/PNMT). A few ChAT-labeled processes were dispersed among TH/PNMT-containing neurons with the majority of overlap immediately ventral to the nucleus ambiguus. By electron microscopy, ChAT-immunoreactivity (ChAT-I) was detected in neuronal perikarya, dendrites, axons and axon terminals and in the vascular endothelial cells of certain blood vessels. The ChAT-labeled perikarya in the ventromedial RVL were medium-sized (15–20 μm), elongated, contained abundant cytoplasm and had slightly indented nuclei. Synaptic junctions on ChAT-immunoreactive perikarya and dendrites were primarily symmetric with 64% (45 out of 70) of the presynaptic terminals unlabeled. The remaining terminals were immunoreactive for ChAT (30%) or TH/PNMT (6%). Terminals with ChAT-I were large (0.8–2.0 μm) and contained numerous small clear vesicles and 1–2 dense core vesicles. Seventy-seven percent (112 out of 145) of the ChAT-labeled terminals formed symmetric synapses with unlabeled perikarya and dendrites, whereas only 8% were with TH/PNMT-labeled perikarya and dendrites, and 15% were with ChAT-immunoreactive perikarya and dendrites. We conclude (1) that cholinergic neurons in the RVL principally terminate on and receive input from non-catecholaminergic neurons, and (2) that the reported sympathetic activation following application of cholinergic agents to the RVL may be mediated by cholinergic inhibition of local inhibitory interneurons. The observed synapses between ChAT and TH/PNMT-containing neurons suggests that cholinergic and adrenergic neurons additionally may exert a minor reciprocal control on each other and thus may modulate their response to the more abundant input from afferents containing other transmitters.     

Evoked slow muscarinic acetylcholinergic synaptic potentials in rat hippocampal interneurons

The hippocampus receives an extensive cholinergic input from the medial septal nucleus that ramifies throughout all layers and plays a pivotal modulatory role in cognitive function. Although the pharmacological effects of exogenous application of cholinergic agonists have been extensively studied in hippocampal neurons, much less is known about the effects of synaptically released acetylcholine (ACh). In this respect, most studies have focused on the cholinergic afferent input to pyramidal neurons that produces a characteristically slow depolarizing synaptic response mediated by activation of muscarinic ACh receptors (mAChRs). Here we report that cholinergic afferent stimulation also elicits atropine-sensitive synaptic potentials in hippocampal CA1 interneurons but, in contrast to synaptic responses in pyramidal neurons, these are highly diverse in waveform, although can still be classified into five distinct subtypes. The most common response type (i) 64% of cells) consisted of a slow sustained membrane potential depolarization. The other 36% of responses could be subdivided into responses comprising of (ii) a biphasic membrane potential change in which an initial slow hyperpolarization subsequently transforms into a slow depolarization (20%), (iii) a pure, slow hyperpolarization (13%), and (iv) an oscillatory response persisting for several seconds (2%). Interestingly, there were also interneurons totally insensitive to both synaptic and pharmacological cholinergic challenge. Morphological investigation of recorded cells revealed no obvious correlation between responsiveness to cholinergic afferent stimulation and dendritic and axonal arborization. The current study suggests that synaptic release of ACh results in a complex and differential mAChR-mediated modulation of cellular excitability within the hippocampal interneuron population.     

Cholinergic function in cultures of mouse spinal cord neurons

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.     

Morphology, Electrophysiology and Pathophysiology of Supragranular Neurons in Rat Primary Somatosensory Cortex

Intracellularly biocytin-labelled neurons in layers 11/111 of adult rat primary somatosensory cortex were analysed for their morphological and electrophysiological properties and studied for their response pattern to transient hypoxia under in vitro conditions. The largest dendritic region is formed by the basal dendrites, which constitute an average area of 0.06 mm² and which can receive synaptic inputs over horizontal distances of more than 300 μm. The dendritic territories formed by the oblique dendrites situated on the apical trunk and by the apical tuft are much smaller. The spine density is highest on the apical trunk, suggesting that large numbers of excitatory synapses are present in this region of the cell. All neurons revealed intrinsic membrane properties of typical regular spiking cells and received an excitatory and a strong biphasic inhibitory input. Whereas a significant correlation could be detected between the cell's input resistance and soma area, no correlation existed between the cell's total dendritic length and input resistance or membrane time constant/input resistance. Neurons responded to transient hypoxia either with an anoxic hyperpolarization with an apparent reversal potential of -82.4 mV, or with a gradual anoxic depolarization which reversed at -56 mV. Oxygen deprivation caused a significant reduction in the extent of axonal collaterals, whereas dendritic proportions and spine density were unaffected. The present study indicates that the dendritic tree is well preserved under in vitro conditions, whereas axonal connections are diminished by oxygen deprivation. Our results further suggest that certain structural properties correlate with the cellular physiology, but that the cell's morphology does not determine its responsiveness to hypoxia.     

Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla

Understanding the visual pathways of the fly's compound eye has been blocked for decades at the second optic neuropil, the medulla, a two-part relay comprising 10 strata (M1-M10), and the largest neuropil in the fly's brain. Based on the modularity of its composition, and two previous reports, on Golgi-impregnated cell types (Fischbach and Dittrich, Cell Tissue Res.,1989; 258:441-475) and their synaptic circuits in the first neuropil, the lamina, we used serial-section electron microscopy to examine inputs to the distal strata M1-M6. We report the morphology of the reconstructed medulla terminals of five lamina cells, L1-L5, two photoreceptors, R7 and R8, and three neurons, medulla cell T1 and centrifugal cells C2 and C3. The morphology of these conforms closely to previous reports from Golgi impregnation. This fidelity provides assurance that our reconstructions are complete and accurate. Synapses of these terminals broadly localize to the terminal and provide contacts to unidentified targets, mostly medulla cells, as well as sites of connection between the terminals themselves. These reveal that R8 forms contacts upon R7 and thus between these two spectral inputs; that L3 provides input upon both pathways, adding an achromatic input; that the terminal of L5 reciprocally connects to that of L1, thus being synaptic in the medulla despite lacking synapses in the lamina; that the motion-sensing input cells L1 and L2 lack direct interconnection but both receive input from C2 and C3, resembling lamina connections of these cells; and that, as in the lamina, T1 provides no output chemical synapses.     

Effects of tetrahydro-9-aminoacridine on cortical and hippocampal neurons in the rat: an in vivo and in vitro study

The effects of tetrahydro-9-aminoacridine (THA), an anticholinesterase drug, have been studied in the rat both in vivo (cerebral cortex) and in vitro (CA1 field of the hippocampus) and compared with those of physostigmine. In the cerebral cortex THA potentiated the excitatory effect of acetylcholine in most neurons, including cortical neurons recorded from chronic unanesthetized animals. In vitro, THA (but not physostigmine) had a depolarizing, atropine- and tetrodotoxin-insensitive effect. This effect is associated with an increase in membrane resistance which suggests a direct effect of THA on hippocampal neurons. In addition THA blocked the slow inhibitory postsynaptic potential. At the same concentration THA potentiated the slow cholinergic excitatory postsynaptic potential produced by electrical stimulation of the cholinergic afferents. Its potency was, however, about 10 times lower than that of physostigmine. These results show that THA: (1) is an anticholinesterase much less potent than physostigmine; but (2) has also direct effects on central neurons, not observed with physostigmine and unrelated to its anticholinesterase activity.     

Synaptic organization in the lamina of the superposition eye of a skipper butterfly, Parnara guttata.

The first optic neuropil of the compound eye, the lamina, of the skipper butterfly Parnara guttata, was examined by light microscopy after Golgi-impregnation and by electron microscopy (EM) to clarify the cellular and synaptic organization. In the lamina, five different types of lamina neurons (L neurons) were characterized by using Golgi-impregnation. By EM, each cartridge was found to contain all nine receptor axons from an ommatidium, five L neurons, and a few putative centrifugal elements. Axons from photoreceptors (retinula cells) R2, R3, R4, R6, R7, and R8 terminate as short visual fibers (svfs) in the lamina cartridge. Those from R1, R5, and R9 penetrate the lamina and terminate in the medulla as long visual fibers (lvfs). In the cartridges, the synaptic contacts were formed from svfs onto L neurons, from the lvfs of R1 and/or R5 to the lvf of R9 and L neurons, and from the lvf of R9 to L neurons. The putative centrifugal fibers also make synapses to svfs and L neurons. At the most distal level of the cartridge, one of the centrifugal fibers containing dense-core vesicles makes presynaptic contacts to the putative long collaterals of the L neuron. A novel characteristic feature of this lamina is that svfs of R3 and R7 and the lvfs of R1 or R5 have long collaterals extending into neighboring cartridges. Presynaptic contacts were confirmed in such long collaterals from the svf. These results imply that receptor axons provide direct intercartridge connections as well as providing indirect connections to neighboring cartridges by way of their input upon L neurons.     

Corticofugal influences on the reticulospinal neurons of gigantocellular nucleus in cats]

Stimulation of the motor cortex evoked excitatory and inhibitory PSP in reticulospinal neurons of the cat gigantocellular nucleus. EPSP were recorded in 94.3% of the investigated neurons and IPSP in 5.7%. Analysis of the presynaptic pathways showed that 77.4% of EPSPs appeared through monosynaptic and 22.6% through polysynaptic corticoreticular connections. According to latency, duration and rise time all monosynaptic EPSP were divided into two groups (fast and slow). Obviously, fast EPSPs are generated by fast corticobulbar fibres and slow ones by slow fibres. IPSP were recorded in neurons which were also inhibited by stimulation of the ventral funiculi of the spinal cord. It is suggested that motor cortical signals can be transmitted to the spinal cord through both mono- and polysynaptic connections of the fast and slow pyramidal neurons with reticulospinal neurons.     

Sensory deprivation changes the pattern of synaptic connectivity in rat barrel cortex

We examined whether sensory deprivation during formation of the cortical circuitry influences the pattern of intracortical single-cell connections in rat barrel cortex. Excitatory postsynaptic potentials from layer 5 pyramidal neurons were recorded in vitro using patch-clamp techniques. In order to evoke such postsynaptic potentials presumptive presynaptic neurons were stimulated by photolytically applied glutamate thus generating action potentials. Synaptic connections between the stimulated and the recorded neuron were identified by the occurrence of postsynaptic potentials following photostimulation. Sensory deprivation altered the projections from layer 2/3 neurons to layer 5 pyramidal cells (L2/3-->L5 projections). In slices of non-deprived rats the input probability of L2/3-->L5 projections showed a periodic pattern with more synaptic connections originating from the borders of the barrel columns, and less synaptic connections originating from the centres. After whisker clipping this periodic pattern disappeared completely and the input probability declined monotonically with increasing distance between stimulated and recorded neuron. These results indicate that sensory input is a prerequisite to establish a synaptic projection pattern which is correlated to the columnar organisation of the anatomical barrel structure.