Ultrastructure of the synapses of the initial axonal segment of the pyramidal neuron

Ultrastructure of the proximal part of the axon in the neurons, identified according to a number of morphological signs as pyramidal, has been studied in the layer III of the cat cerebral hemisphere sensomotor cortex. In sections, tangential to the cortical surface, in the initial axonal segment, a submembranous osmophilic layer and fasciculi of microtubules are revealed. On the initial segment spines are found, they contain cysterns resembling by their structure the spine system of the dendritic spines. Axonal terminals revealed along the axonal distribution are in contact both with the axonal trunk and with the spines. Regarding the initial segment, they are presynaptic, contain oval synaptic vesicles and form symmetric axo-axonal synapses only. In transversal sections axonal terminals are detected, arranging on the surface of the initial segment mostly as single ones, in longitudinal sections they are seen as clusters. Analysing the author's data and those from the literature, a conclusion is made that in intact animals the synaptic contacts at the initial segment of the axon are the only form of axo-axonal synapses in the neocortex.     

Morphological characteristics of various segments of local connections in the rat somatosensory cortex

Pyramidal, aspinous, sparsely-spinous bipolar and multipolar neurons of the rat sensomotor cerebral cortex, impregnated after Golgi method, have been studied at an electron microscopical level. The ultrastructural characteristics of the pyramidal neurons differs from that of the nonpyramidal cells. Distribution of various synaptic contacts on the cellular surface and cortical postsynaptic targets of the axonal arborizations of the neurons are revealed. On the body of the pyramidal cells only symmetrical synapses exist, on large dendritic trunks symmetrical synapses prevail, on the spines and the terminal dendritic branches assymetrical synapses mainly predominate. Axonal collateralies of the pyramidal cells form asymmetrical synapses on the spines, small and middle dendrites. There are more axo-somatic synapses on the bodies of the nonpyramidal neurons than on the pyramidal cells, among them both symmetrical and asymmetrical types of the synapses occur. On the trunks and small dendrites of the nonpyramidal cells both types of synaptic contacts are revealed. In the distal direction of the dendrites the number of the asymmetrical synapses becomes predominating. Axons of the bipolar cells form asymmetrical synapses on the spines, small and middle dendrites. Axons of the multipolar cells form symmetrical synapses on the dendrites and the dendritic trunks of the nondifferentiated cells. Differences in the distribution character of the synaptic inlets and various postsynaptic targets of the axonal systems in the cells assume various functional role of the identified neurons.     

A light and electron microscopic study of betaine/GABA transporter distribution in the monkey cerebral neocortex and hippocampus

The present study aimed to elucidate the distribution of betaine/γ-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.     

Synaptic localization of adrenergic disinhibition in the rat hippocampus.

Norepinephrine is an endogenous neurotransmitter that reduces synaptic inhibition onto pyramidal neurons in the hippocampus by an action at an alpha-adrenergic receptor. The physiological mechanism of this disinhibition was previously not known, except that it occurred at a site presynaptic to the inhibited pyramidal cell. In this paper we present evidence that adrenergic disinhibition is restricted to the early phase of the evoked inhibitory postsynaptic potential in area CA1 of the hippocampus. The locus of disinhibition does not appear to reside in the interneuronal terminal, axon, or cell body. Instead, adrenergic agonists appear to reduce evoked synaptic inhibition by depressing excitatory synapses that activate the interneuron.     

Anatomical circuitry of lateral inhibition in the eye of the horseshoe crab, Limulus polyphemus

Lengthy uninterrupted series of sections of the neural plexus in the compound eye of the horseshoe crab, Limulus polyphemus, have been used to reconstruct all the arborizations and their synaptic interconnections in a neuropil knot. This one microglomerulus contains the axons of 19 retinular cells, which pass by without contacts; 13 efferent fibres with 44 synapses to and from eccentric cell collaterals; and arborizations from 54 eccentric cells with 577 synapses. Eccentric cell axons are devoid of synaptic input. Their collaterals ramify in synaptic knots and subserve both pre- and postsynaptic functions simultaneously. Arborizations near the axon of origin have a highly branched pattern (up to 20 bifurcations), a high synaptic input: output ratio (up to about 9:1), and high synaptic density (a maximum of 12 per micrometre of neurite length). The opposite extreme is represented by sparsely branched eccentric cell collaterals distant from their axons of origin with very little synaptic input and sparse output. Spatially graded lateral inhibition is the apparent outcome of a radially decreasing distribution of inhibitory synapses on the arborizations of eccentric cell collaterals combined with possible decremental signal transmission in the plexus. The synaptic analysis has a bearing on most physiological aspects of lateral inhibition that have been studied in the Limulus eye. Implied in the results is the suggestion that synapse formation is an intrinsic property of the presynaptic element, but that the connectivity is governed by the electrical activity of target neurons.     

Characterization of orexin A immunoreactivity in the rat area postrema

The distribution of orexin A immunoreactivity and the synaptic relationships of orexin A-positive neurons in the rat area postrema were studied using both light and electron microscopy techniques. At the light microscope level, numerous orexin A-like immunoreactive fibers were found within the area postrema. Using electron microscopy, immunoreactivity within fibers was confined primarily to the axon terminals, most of which contained dense-cored vesicles. Both axo-somatic and axo-dendritic synapses made by orexin A-like immunoreactive axon terminals were found, with these synapses being both symmetric and asymmetric in form. Orexin A-like immunoreactive axon terminals could be found presynaptic to two different immunonegative profiles including the perikarya and dendrites. Occasionally, some orexin A-like immunoreactive profiles, most likely to be dendrites, could be seen receiving synaptic inputs from immunonegative or immunopositive axon terminals. The present results suggest that the physiological function of orexin A in the area postrema depends on synaptic relationships with other immunopositive and immunonegative neurons, with the action of orexin A mediated via a self-modulation feedback mechanism.     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.     

The wiring-in of neural nets revisited

Earlier and some recent ideas about the possible modes of specification of the wiring-in of nervous systems are reviewed in the light of older and several recent experiments, and some new ideas are suggested. It is argued that certain general principles, notably the postulated ‘principle of alternative matching’ (PALMA) and a suggested and related ‘kaleidoscopic effect’ (KALEF), as well as the notion of an ‘extracellular guidance network’ (ECGN), are in good agreement with recent and older findings concerning axonal guidance during neural wiring-in. It seems possible that by means of genetically programmed processes, neurons become systematically combinatorially labelled to such a degree that possibly all neurons areuniquely specified, as regards the combination oftypes of cell labels they make. Yet, there remains considerable freedom as regards the modes of arrangements of cell labels within cell surface membranes and the KALEF permits to overcome apparent difficulties that confronted earlier versions of the cell labelling hypotheses (cf. Edelman,Science 219, 450–457, 1983, for mention of such difficulties). Apart from label specification, neural development seems to depend on trophic factors, which are also essential for the maintenance of the developed nervous system. The systematic programmes for cell labelling, apart from generating all the required neurons, also produces inappropriate neurons and synaptic connections. These are got rid of by systematic cell death and/or atrophy of inappropriate synapses and/or elimination of inappropriate axon collaterals. The resulting neural net seems then very specifically wired-in for each species, apparently without redundant neurons.     

Electron microscopy examination of galanin-like peptide (GALP)-containing neurons in the rat hypothalamus

Galanin-like peptide (GALP) is a novel peptide which is isolated from the porcine hypothalamus. GALP-containing neurons are present in the arcuate nucleus (ARC), being particularly densely concentrated in medial posterior regions. To observe the ultrastructure and synaptic relationships of GALP-containing neurons in the ARC, light and immunoelectron microscopy techniques were used. At the light microscope level, GALP-containing neurons were observed distributed rostrocaudally throughout the ARC, with the majority present in the posterior, periventricular zones. At the electron microscope level, many immunopositive dense-cored vesicles were evident in the perikarya, dendrites and axon terminals of the GALP-containing neurons. Furthermore, these neurons received synapses from immunonegative axon terminals that were symmetric in the case of synapses made on perikarya, and both asymmetric and symmetric for synapses made on dendrites. Axon terminals of GALP-containing neurons often made synapses on immunonegative dendrites. Such synapses were all symmetric. Synapses were also found between axon terminals and perikarya as well as dendrites of GALP-containing neurons. These findings suggest that the physiological role of the GALP-containing neurons in the ARC is based on complex synaptic relationships between GALP-containing neurons and either GALP-immunopositive or -immunonegative neurons.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.     

Glycine and serine as tentative agonists for metabotropic glutamate receptors

In experiments on slices of the rat hippocampus, glycine (Gly) and serine (Ser) in concentrations of 100 μM to I mM were found to reversible increase the amplitudes of population EPSP (pEPSP) in pyramidal neurons of theCA1 hippocampal area, evoked by single electrical stimuli applied to Schaffer collaterals (SchC). This potentiation was not affected by 100 μM of a non-competetive antagonist of NMDA glutamate receptors (GR), ketamine, but was considerably weakened by 500 μM of a competitive antagonist of metabotropic GR (mGR), (±)-4-carboxyphenylglycine (CFG). The effects of Gly and Ser were not observed in the presence of 50 μM of a blocker of protein kinase C (PKC) catalytic subunit, polymixin B, but were not modified by preliminary action on the slices of 10 μM of a calmodulin inhibitor, substance W-7. Gly and Ser also enhanced long-term post-tetanic potentiation (LTPP) of synaptic transmission caused by high-frequency rhythmic stimulation of SchC. Low-frequency (1/sec, 15 min) SchC stimulation abolished the potentiation of synaptic transmission evoked either by high-frequency SchC stimulation or by the actions of Gly and Ser. The data allow us to suggest that Gly and Ser in millimolar concentrations activate mGR, enhance relay functions of the synapses of pyramidal neurons in theCA1 hippocampal region, and facilitate plastic modifications in these synapses.     

Ultrastructural characteristics of layer III pyramidal neurons in the cat primary auditory cortex (Al)

Electron microscope studies were made of retrogradely horseradish peroxidase-labeled pyramidal neurons forming transcallosal projections in layer III of the cat primary auditory cortex (Al). These showed a significant proportion of the somatic membrane to be covered with processes of astroglia, while synapses occupy 20% of the synaptic surface on average. Between 4 and 10 axosomatic synapses were identified on the profiles of callosal cell somata. All these were formed by axonal terminals containing small, flattened synaptic vesicles and had symmetrical contacts. Average length of these synaptic contacts equaled 1.6 µm. Numerous anterogradely horseradish peroxidase-labeled axonal terminals of callosal fibers were found in cortical area Al in amongst retrogradely HP-labeled neurons. The ultrastructural pattern of these is described.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 520–526, July–August, 1990.     

Role of the adenylate cyclase system in cholinergic modulation of synaptic transmission in the hippocampus

Involvement of the adenylate cyclase system in cholinergic modulation of synaptic transmission was investigated in area CA1 in rat hippocampal slices. Microiontophoretic application of acetylcholine as well as addition of carbachol to the superfusate or of tolbutamide (a cAMP-dependent protein kinase inhibitor) depressed transmission at synapses formed by Schaffer collaterals and commissural fibers with dendrites of pyramidal cells belonging to hippocampal area CA1. Both numbers of free quanta of neurotransmitter and the likelihood of transmitter release decreased following carbachol action. Atropine suppressed the inhibitory action of carbachol on synaptic transmission. Dibutyryl cAMP and forskolin increased the amplitude of synaptic potentials and suppressed, either partially or in full, the inhibitory effects of cholinomimetics on synaptic potentials. It was concluded that cholinomimetics and activators of the adenylate cyclase system exert opposing effects on neurotransmission at synapses formed between Schaffer collaterals/commissural fibers and dendrites of pyramidal neurons belonging to hippocampal area CA1.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 435–442, July–August, 1989.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Glutamatergic components of the retrosplenial granular cortex in the rat

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 μm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V–VI; some were also present in layers I–III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.     

Electron microscope study of sources in the cat primary auditory cortex (AI) projecting to the medial geniculate body

An electron microscope study of retrogradely labeled pyramidal neurons in layer VI of the primary auditory cortex (AI) after injecting horseradish peroxidase (HP) into the medial geniculate body was carried out in cats. Not less than 57.8±1.9% on average of the perimeter of perikaryon profiles of corticogeniculate neurons labeled with HP were found to be covered with astroglia processes. Between three and eight synapses occupying an average of 10.8±1.0% of the perimeter length were found on the perikaryon profiles of these neurons. Nearly all synapses (a total of 98.7%) at the soma of corticogeniculate neurons had symmetrical active zones, being made up of axonal terminals with flattened synaptic vesicles. Anterogradely HP-labeled axonal terminals of geniculocortical fibers were also found in the neuropil of layer VI in area AI, in addition to retrogradely labeled neurons. They contained large round synaptic vesicles and formed asymmetrical synapses. The potential role of axosomatic synapses in the shaping of corticogeniculate neuronal activity is discussed.A. A. Bogomolets Institute, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 171–178, March–April, 1990.     

Ultrastructural alterations in the initial segments and in the recurrent collateral terminals of Purkinje cells following axotomy

Summary Transection of Purkinje cell axons in adult male rats made 1.5 mm or further from the cell body does not lead to the death of the neuron and results in compensatory structural alterations of the surviving axonal portions of the nerve cell. Near to, and at the emergence of recurrent collaterals of Purkinje cell axons, huge varicosities filled with filaments, granular material, lysosomes and mitochondria develop. Terminals of recurrent axon collaterals also exhibit different degrees of structural changes. Most striking of the morphological alterations is the regular presence of nematosomes in the hypertrophic axonal branches, especially in synaptic terminals. Since nematosomes were shown to contain RNA in other types of neurons, their presence in recurrent collaterals may indicate an enhanced synthetic activity in Purkinje axonal processes and endings after axotomy.