Developmental down-regulation of GAP-43 expression and timing of target contact in rat corticospinal neurons

During early nervous system development axons grow toward the target tissue that they will innervate. As axons invade target tissue, growth slows and ceases. Neurons express high levels of the growth-associated protein GAP-43 during developmental axon growth, declining with maturation. It has been suggested that target contact provides a signal which down-regulates GAP-43 expression. To study this issue in more detail, we used in situ hybridization to quantify relative changes in GAP-43 mRNA in corticospinal tract neurons identified by Fast Blue retrograde labeling. We also used anterograde transport of biotinylated dextran amine to study the invasion of target by corticospinal axons. We find that GAP-43 mRNA is high during the first postnatal week and then declines in two phases. Approximately half of the initial level of GAP-43 expression in corticospinal neurons is lost by P12; then expression remains at a plateau until P21. Between P21 and P28, GAP-43 expression again declines by half and then remains steady at the adult level (one fourth of initial level). Corticospinal axons initially invade spinal gray matter during the first 2 postnatal weeks, in a rostrocaudal gradient. Varicosities suggestive of terminal boutons become numerous during the third and fourth week, and the morphology of corticospinal axon terminals achieves the mature form at the end of the fourth week. These data suggest that the first phase of down-regulation of GAP-43 in corticospinal neurons is coincident with initial target contact and that the second phase is coincident with final maturation of terminal arborization.     

Preferential localization of the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are involved in the control of neuronal excitability and plasticity. In this study, we used immunoblotting and immunohistochemical techniques to reveal the developmental expression and subcellular distribution of the HCN1 subunit in the cerebellar cortex. During postnatal development, the spatio-temporal expression of HCN1 correlated well with the morphological events occurring during the ontogenesis of cerebellar interneurons. Using immunoblotting techniques, HCN1 was weakly detected during the first postnatal week and continued to increase throughout postnatal development, peaking at postnatal day (P)15. At the light-microscopic level, HCN1 immunoreactivity was very weak until P7 whereas from P10-12 to adulthood it was strongly detected in the lower third of the molecular layer and in the Purkinje cell layer. HCN1 was present in axons running through the molecular layer and in the pericellular basket around Purkinje cells at P12, but in the periaxonal plexus (the pinceau) surrounding their initial segment only after P15. Using immunofluorescence, HCN1 colocalized with GAD65 and synaptophysin, demonstrating that the subunit was present in inhibitory axons and axon terminals. At the electron-microscopic level, in adulthood, HCN1 immunoparticles were detected at postsynaptic sites in basket and Purkinje cells but most immunoparticles were found at presynaptic sites in basket cell axons and in terminals. In the axon terminals, the distribution of HCN1 was relatively uniform along the extrasynaptic plasma membrane; this was confirmed using quantitative techniques. The present findings suggest that HCN1 channels may provide a significant route for modulating co-ordinated cerebellar synaptic transmission through basket cells.     

Differential distribution and regulation of galanin receptors- 1 and -2 in the rat lumbar spinal cord

The proper function of the brain depends on a precise arrangement of excitatory and inhibitory synapses. Although the caudal nucleus of tractus solitarius (cNTS) plays a pivotal role in cardiorespiratory reflexes, we know little about the formation of the local neural network in the cNTS. In the present study, we have focused on GABAergic axon terminals and investigated postnatal changes in GABAergic synaptic organizations in the rat cNTS immunocytochemically at both light and electron microscopic levels. Counting synaptic and non-synaptic GABAergic axon terminals revealed that GABAergic axon terminal number in the cNTS seemed constant until the second postnatal week and that GABAergic axon terminals were reorganized around postnatal day 10 (P10). Electron microscopic observation revealed that more than 20% GABAergic axon terminals formed axosomatic synapses at P2 to P4, but the number of GABAergic axosomatic synapse on neurons with smaller soma (smaller neurons) decreased considerably after P8. Orphan GABAergic boutons were present around somata of smaller neurons at P10, and axodendritic synapse number on thicker dendrites decreased gradually during postnatal development. These results show that GABAergic axon terminals detach from somata of smaller neurons at the second postnatal week. Such morphologic changes in axon terminals could cause changes in electrophysiological activity and might contribute to reorganization of the local network within the cNTS from neonatal to adult type. These postnatal changes in the cNTS local network might be prerequisite for the cardiorespiratory reflexes of the adult type.     

Morphological development and maturation of the GABAergic synapses in the mouse cerebellar granular layer

In the adult central nervous system (CNS), gamma-amino butyric acid (GABA) is a predominant inhibitory neurotransmitter, which regulates glutamatergic activity. Recent studies have revealed that GABA serves as an excitatory transmitter in the immature CNS, and is involved in brain morphogenesis. To elucidate how GABA exerts its effect on immature neurons and how GABAergic synapses are formed, we examined both development of pre- and post-synaptic elements of the GABAergic synapses formed between granule and Golgi cells in the mouse cerebellar granular layer. Immunohistochemistry for glutamic acid decarboxylase (GAD) demonstrated that GABA was localized throughout the Golgi cells before postnatal day 7 (P7), and became confined to the axon terminals during the second postnatal week. Electron microscopic analysis demonstrated that GABAergic synapses were clearly detected at P10. In situ hybridization and immunohistochemistry for the GABA(A) receptor alpha1 and alpha6 subunits, which are mainly involved in inhibitory synaptic transmission, demonstrated that both subunits appeared at P7. Distribution of both subunits expanded in the granular layer with special reference to the development of GABAergic synapses. Furthermore, the majority of the subunits accumulated adjacent to the GABAergic terminals. These results suggested that in the granular layer, GABA might be non-synaptically secreted from Golgi cell axons and dendrites during the first postnatal week. From the second postnatal week, GABA is synaptically released and begins to mediate inhibitory transmission. Furthermore, it was suggested that GABAergic innervation could initiate expression and trafficking of the GABA(A) receptors containing the alpha1 and alpha6 subunits.     

A quantitative analysis of axon outgrowth, axon loss, and myelination in the rat pyramidal tract

A quantitative analysis of the development of the pyramidal tract (PT) was carried out at the level of the caudal medulla oblongata and at the sixth cervical spinal segment (C6), in rats ranging in age from embryonic day 20 (E20) to the adult of 90 days postnatally (P90). The axon number in the right medullary PT rises from 27,000 axons at E20 to 391,000 axons at P4. Growth cones are abundant during this period, but can still be observed occasionally at P7. After P4, the axon number is reduced by 62%, to 150,000 in the adult. A rapid axon loss until P14 is followed by a gradual axon loss, continuing beyond the third postnatal week. A similar biphasic axon loss was observed in the cervical PT. At P2 and at P7, concentrations of electron-dense material were observed in 0.5-0.7% of the axon profiles in the medullary PT. Since at P21 this feature was only observed in 0.2% of the axons, it might represent an early sign of axon loss. Myelination starts in the medullary PT at P7. Especially during the third postnatal week, the number of myelinated axons increases rapidly. In the adult rat PT, both at medullary and cervical levels, about one third of the axons are still unmyelinated. The results indicate that the development of the rat PT is characterized by a gradual outgrowth of its fibers and by a protracted, biphasic axon loss. Furthermore, comparing the PT at the medulla, at C3, and at C6, a rostrocaudal decrease in axon number was observed during development as well as at the adult stage. Therefore, no evidence was found for increased axon branching in the tract in the cervical intumescence.     

High number of striatal dopaminergic neurons during early postnatal development: correlation analysis with dopaminergic fibers

Dopamine (DA) axons in the developing striatum cluster in discrete areas called "DA islands". During the third postnatal week, most DA islands are no-longer detectable and the DA innervation becomes uniform. In this study we explored the relationship between the pattern of DA innervation and the number of striatal tyrosine hydroxylase positive (TH+) cells during early postnatal development. By using dedicated stereology we found that the newborn striatum contains striatal TH+ cells, which cluster around newly sprouted DA axons. The number of these cells decreases when DA axons develop a full pattern of striatal innervation. This condition suggests a causal relationship between the amount of striatal DA innervation and the presence of striatal DA neurons. A better knowledge of the mechanisms regulating the ontogenesis of the nigrostriatal DA system may pave the way to strategies of neurorescue of the DA system.     

Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient

Vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 show complementary distribution in neocortex; VGLUT1 is expressed mainly in axon terminals of neocortical neurons, whereas VGLUT2 is located chiefly in thalamocortical axon terminals. However, we recently reported a frequent colocalization of VGLUT1 and VGLUT2 at a subset of axon terminals in postnatal developing neocortex. We here quantified the frequency of colocalization between VGLUT1 and VGLUT2 immunoreactivities at single axon terminals by using the correlation coefficient (CC) as an indicator in order to determine the time course and spatial extent of the colocalization during postnatal development of mouse neocortex. The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood. CC in layer IV showed a peak at P7 in area S1, and at P10 in area V1 though the latter peak was much smaller than the former. These results suggest that thalamocortical axon terminals contained not only VGLUT2 but also VGLUT1, especially at P7-10. Double fluorescence in situ hybridization confirmed coexpression of VGLUT1 and VGLUT2 mRNAs at P7 in the somatosensory thalamic nuclei and later in the thalamic dorsal lateral geniculate nucleus. As VGLUT1 is often used in axon terminals that show synaptic plasticity in adult brain, the present findings suggest that VGLUT1 is used in thalamocortical axons transiently during the postnatal period when plasticity is required.     

Development of striatal compartmentalization following pre- or postnatal dopamine depletion.

Nigrostriatal dopamine (DA) projections terminate in distinct patches during the late prenatal and early postnatal period in the rat. During the first postnatal week, patches of DA fibers overlap with clusters of striatal neurons that share several identified characteristics. The early segregation of striatal cell types into either these patches or the surrounding matrix becomes a permanent organizational feature of the striatum. In order to determine whether the heterogeneous distribution of DA influences the formation of cellular patches, the developmental organization of chemically identifiable cell types was examined in normal rats and in rats DA depleted as infants (0 or 3 d) or in utero (embryonic days 17-18). During the first postnatal week, corresponding patches of DA afferents and substance P (SP)-immunoreactive neurons existed in the striatum of normal animals, and AChE-positive zones overlapped these patches in the lateral striatum. Injection of 6-hydroxydopamine into the lateral ventricles of fetal or infant rats produced a dramatic loss of striatal DA terminals. Neither the patchy distribution of SP-immunoreactive neurons nor the distinctive pattern of AChE staining present during the first 2 postnatal weeks was disrupted. During the third postnatal week, cells immunoreactive for leu-enkephalin or calbindin-D28k were confined to the matrix compartment, and this compartmentalization was also not noticeably changed by pre- or postnatal DA depletion. In adult animals, overlapping patches of leu-enkephalin- and SP-immunoreactive fibers were observed, regardless of whether any DA terminals remained. Thus, the basic organization of the striatal patch and matrix compartments develops normally in the absence of DA innervation through much of the formative period. Although these observations do not completely dismiss the possibility that the first DA afferents to appear in the striatal primordia influence contracted striatal cells to develop the patch phenotype, they suggest that the patchy distribution of DA afferents may be secondary to the early clustering of striatal neurons forming the patch compartment.     

Organization and regulation of paraventricular nucleus glutamate signaling systems: N-methyl-D-aspartate receptors

The initial pattern of corticospinal (CS) terminations, as axons grow into the spinal gray matter, bears little resemblance to the pattern later in development and in maturity. This is because of extensive axon pruning and local axon terminal growth during early postnatal development. Pruning is driven by activity-dependent competition between the CS systems on each side during postnatal weeks (PW) 3-7. It is not known whether CS axon terminal growth and final topography are activity dependent. We examined the activity dependence of CS axon terminal growth and topography at different postnatal times. We inactivated sensory-motor cortex by infusion of the gamma-aminobutyric acid type A (GABA(A)) agonist muscimol and traced CS axons from the inactivated side. Inactivation between PW5 and PW7 produced permanent changes in projection topography, reduced local axon branching, and prevented development of dense clusters of presynaptic sites, which are normally characteristic of CS terminals. Inactivation at younger (PW3-5) and older (PW8-12) ages did not affect projection topography but impeded development of local axon branching and presynaptic site clusters. These effects were not due to increased cortical cell death during inactivation. Neural activity plays an important role in determining the morphology of CS terminals during the entire period of development, but, for the projection topography, the role of activity is exercised during a very brief period. This points to a complex, and possibly independent, regulation of termination topography and terminal morphology. Surprisingly, when a CS neuron's activity is blocked during early development, it does not recover lost connections later in development once activity resumes.     

Maturation of rat visual cortex. III. Postnatal morphogenesis and synaptogenesis of local circuit neurons

The postnatal development of 3 types of local circuit neurons in rat visual cortex was examined in Golgi and electron microscopic preparations. During the first postnatal week, smooth and sparsely spinous stellate, bitufted and bipolar neurons were identified in Golgi material by their characteristic dendritic arborizations. Morphological differentiation begins during this week, as each neuron sprouts dendrites which extend, branch and produce spines, and ends by day 21. This differentiation was traced by quantifying the somatic area and number of primary dendrites on stellate, bitufted and bipolar neurons in layer II/III or layer V. Neurons in deep cortex differentiate earlier than those in superficial laminae. On day 3, axons are evident as short, straight processes, however, by day 6, many axons have branches and varicosities. The increase in the complexity of the axonal trees continues during the second and third postnatal weeks. Since the axons of stellate and bitufted neurons form synapses with the somata of pyramidal neurons, an index of the synaptogenesis of these neurons was traced by counting the numbers of synapses on the somata of pyramidal neurons. The mean number of axosomatic synapses increases steadily from day 3 to day 30. Layer V pyramidal neurons form axosomatic synapses before pyramidal neurons in layer II/III. In conclusion, the morphology of local circuit neurons develops during the period after they migrate into cortex. The principle that cortical local circuit neurons develop after projection neurons only applies for the synaptogenesis of the axon, but not for the maturation of the cell body and dendrites.     

Polarised Localisation of the Voltage-Gated Sodium Channel Nav1.2 in Cerebellar Granule Cells

Voltage-gated sodium channels are responsible for action potential initiation and propagation in electrically excitable cells. In this study, we used biochemical, immunohistochemical and quantitative immunoelectron microscopy techniques to reveal the temporal and spatial expression of the Na_v1.2 channel subunit in granule cells of cerebellum. Using histoblot, we detected Na_v1.2 widely distributed in the adult brain, but prominently expressed in the cerebellum. During postnatal development, Na_v1.2 mRNA and protein were detected low during the first and second postnatal week, increased to P15 and then continue to decrease until adult levels. At the light microscopic level, Na_v1.2 immunoreactivity concentrated in the molecular layer of the cerebellar cortex. Using immunofluorescence, Na_v1.2 colocalised with VGluT1, but not with VGluT2, demonstrating that the subunit was preferentially present in parallel fibre axons and axon terminals. At the electron microscopic level, Na_v1.2 immunoparticles were exclusively detected at presynaptic sites in granule cell axons and axon terminals of granule cells, with occasional clustering in their axon initial segment. This was demonstrated using quantitative immunogold analysis. In the axon terminals, the distribution of Na_v1.2 was relatively uniform along the extrasynaptic plasma membrane and never detected in the active zone. We could not find detectable levels of Na_v1.2 at postsynaptic elements of granule cells or other cerebellar cell types. The present findings show a polarised distribution of Na_v1.2 along the neuronal surface of granule cells and suggest its primary involvement in the transmission of information from granule cells to Purkinje cells.     

Postnatal changes in arborization patterns of murine retinocollicular axons

The growth and arborization of murine retinocollicular axons have been studied by means of HRP axon filling during postnatal development. Transformations in arborization patterns have been correlated with changes in synaptic density in the superficial collicular neuropil and with the formation of synapses by HRP-filled axons. At all postnatal ages axons of the optic projection are fasciculated and most follow a rostrocaudally aligned path. On the day of birth the axons course through both stratum griseum superficiale (SGS) and stratum opticum (SO); during the following 4 days the axon trunks disappear from SGS and are subsequently found only in SO. From postnatal day (P) 0 to P3, the majority continue far caudally in the colliculus, giving rise to small ascending collaterals at multiple points along their course. Ultimately, usually by P3, one or two collaterals begin to branch profusely and by P5 the majority of axons give rise to a focal terminal ascending arborization. The general configuration of most arborizations at P3 approximates that of the mature axon. However, the richness of terminal branching increases from P3 through the first 2 postnatal weeks. Synaptic density is relatively low in the first postnatal week, and no synapses involving HRP-filled optic axons were identified in this interval. Subsequently, after elaboration of definitive arbors has begun, synaptogenesis in the surrounding neuropil accelerates. Synaptic density in the upper SGS approximates adult values early in the third postnatal week. By this time synaptic junctions involving the terminal arborizations of optic axons are abundant.     

Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain

Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single- and double-immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2-loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal-like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate-putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2-loaded glutamatergic axons maturate earlier than VGluT1-laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood.     

Developmental expression of GABA transporter-1 and 3 during formation of the GABAergic synapses in the mouse cerebellar cortex

In the brain, gamma-amino butyric acid (GABA), released extrasynaptically and synaptically from GABAergic neurons, plays important roles in morphogenesis, expression of higher functions and so on. In the GABAergic transmission system, plasma membrane GABA transporters (GATs) mediate GABA-uptake from the synaptic cleft in the mature brain and are thought to mediate diacrine of cytosolic GABA in the immature brain. In the present study, we focused on two GATs (GAT-1 and GAT-3) in the mouse cerebellar cortex, which are widely localized in neural and glial cells. Firstly, we examined the localization of GATs in the dendrites and cell bodies of developing GABAergic neurons, where GABA is extrasynaptically distributed, to clarify the GABA-diacrine before synaptogenesis. Secondly, we examined the developmental changes in the localization of GATs to reveal the development of the GABA-uptake system. Neither transporter was detected within the dendrites and cell bodies of GABAergic neurons, including Purkinje, stellate, basket and Golgi cells, in the immature cerebellar cortex. GAT-1 was observed within the Golgi cell axon terminals after postnatal day 5 (P5) and presynaptic axons of stellate and basket cells after P7. GAT-3 was localized within the astrocyte processes, sealing the GABAergic synapses in the Purkinje cell and granular layers after P10. These results indicated that GABA-diacrine did not work in the mouse cerebellar cortex. The onset of GAT-1-expression was prior to that of GAT-3. GAT-1 started to be localized within the GABAergic axon terminals during synapse formation. GAT-3 started to be localized within astrocyte processes when they sealed the synapses.     

Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons

Despite the increasing use of genetically modified mice to investigate the dopamine (DA) system, little is known about the ultrastructural features of the striatal DA innervation in the mouse. This issue is particularly relevant in view of recent evidence for expression of the vesicular glutamate transporter 2 (VGLUT2) by a subset of mesencephalic DA neurons in mouse as well as rat. We used immuno-electron microscopy to characterize tyrosine hydroxylase (TH)-labeled terminals in the core and shell of nucleus accumbens and the neostriatum of two mouse lines in which the Vglut2 gene was selectively disrupted in DA neurons (cKO), their control littermates, and C57BL/6/J wild-type mice, aged P15 or adult. The three regions were also examined in cKO mice and their controls of both ages after dual TH-VGLUT2 immunolabeling. Irrespective of the region, age and genotype, the TH-immunoreactive varicosities appeared similar in size, vesicular content, percentage with mitochondria, and exceedingly low frequency of synaptic membrane specialization. No dually labeled axon terminals were found at either age in control or in cKO mice. Unless TH and VGLUT2 are segregated in different axon terminals of the same neurons, these results favor the view that the glutamatergic cophenotype of mesencephalic DA neurons is more important during the early development of these neurons than for the establishment of their scarce synaptic connectivity. They also suggest that, in mouse even more than rat, the mesostriatal DA system operates mainly through non-targeted release of DA, diffuse transmission and the maintenance of an ambient DA level.     

Pre- and postsynaptic sites for serotonin modulation of GABA-containing neurons in the shell region of the rat nucleus accumbens

The shell of the nucleus accumbens receives a dense serotonergic innervation and contains abundant gamma-aminobutyric acid (GABA)-immunoreactive neurons. Moreover, serotonin (5-hydroxytryptamine: 5-HT) and GABA have been implicated in a variety of common motivational and motor-related functions partially ascribed to this brain area. We used immunoelectron microscopy of antisera directed against 5-HT and GABA in the same section of tissue to examine whether there were cellular substrates that might indicate more specific sites for functional interactions involving these transmitters in the shell region of the rat nucleus accumbens. Immunogold-silver labeling for GABA was localized to perikarya, dendrites, axons and axon terminals, whereas immunoperoxidase labeling for 5-HT was restricted to axons and axon terminals. Approximately half (187/366) of the 5-HT-immunoreactive axon terminals apposed or formed synaptic junctions with postsynaptic neurons. These junctions were mainly of the symmetric-type (83/187) characteristic of inhibitory transmitters, and were equally prevalent on dendrites with and without detectable gold-silver labeling for GABA. Of the 187 5-HT-labeled axon terminals with recognized synaptic contacts, 36% also showed convergence on a common dendrite with a GABA-labeled axon terminal. In addition, 5-HT- and GABA-immunoreactive axon terminals were commonly (83/366) identified in direct apposition to one another. Within a single plane of section, 41% of the apposed GABA-immunoreactive axon terminals formed symmetric-type junctions with dendrites or somata, whereas, the apposed 5-HT-labeled axon terminals rarely showed postsynaptic contacts. These results indicate that 5-HT-containing axon terminals may postsynaptically inhibit GABAergic neurons and their targets within the shell of the rat nucleus accumbens. Additionally, our results strongly suggest that, in this brain region, appositions between 5-HT and GABA axons and axon terminals may facilitate presynaptic interactions between these transmitter systems. © 1996 Wiley-Liss, Inc.     

Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy.

The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.     

Postnatal development of chorda tympani axons in the rat nucleus of the solitary tract

The chorda tympani nerve (CT), one of three nerves that convey gustatory information to the nucleus of the solitary tract (NTS), displays terminal field reorganization after postnatal day 15 in the rat. Aiming to gain insight into mechanisms of this phenomenon, CT axon projection field and terminal morphology in NTS subdivisions were examined using tract tracing, light microscopy, and immunoelectron microscopy at four postnatal ages: P15, P25, P35, and adult. The CT axons that innervated NTS rostrolateral subdivision both in the adult and in P15 rats were morphologically distinct from those that innervated the rostrocentral, gustatory subdivision. In both subdivisions, CT terminals reached morphological maturity before P15. Rostrolateral, but not rostrocentral axons, went through substantial axonal branch elimination after P15. Rostrocentral CT synapses, however, redistribute onto postsynaptic targets in the following weeks. CT terminal preference for GABAergic postsynaptic targets was drastically reduced after P15. Furthermore, CT synapses became a smaller component of the total synaptic input to the rostrocentral NTS after P35. The results underlined that CT axons in rostrocentral and rostrolateral subdivisions represent two distinct populations of CT input, displaying different morphological properties and structural reorganization mechanisms during postnatal development.     

Ultrastructural Evidence for Synaptic Interactions between Thalamocortical Axons and Subplate Neurons

Thalamic axons are known to accumulate in the subplate for a protracted period prior to invading the cortical plate and contacting their ultimate targets, the neurons of layer 4. We have examined the synaptic contacts made by visual and somatosensory thalamic axons during the transition period in which axons begin to leave the subplate and invade the cortical plate in the ferret. We first determined when geniculocortical axons leave the subplate and begin to grow into layer 4 of the visual cortex by injecting 1,1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine (Dil) into the lateral geniculate nucleus (LGN). By birth most LGN axons are still confined to the subplate. Over the next 10 days LGN axons grow into layer 4, but many axons retain axonal branches within the subplate. To establish whether thalamic axons make synaptic contacts within the subplate, the anterograde tracer PHA-L was injected into thalamic nuclei of neonatal ferrets between postnatal day 3 and 12 to label thalamic axons at the electron microscope level. The analysis of the PHA-L injections confirmed the Dil data regarding the timing of ingrowth of thalamic axons into the cortical plate. At the electron microscope level, PHA-L-labelled axons were found to form synaptic contacts in the subplate. The thalamic axon terminals were presynaptic primarily to dendritic shafts and dendritic spines. Between postnatal days 12 and 20 labelled synapses were also observed within layer 4 of the cortex. The ultrastructural appearance of the synapses did not differ significantly in the subplate and cortical plate, with regard to type of postsynaptic profiles, length of postsynaptic density or presynaptic terminal size. These observations provide direct evidence that thalamocortical axons make synaptic contacts with subplate neurons, the only cell type within the subplate possessing mature dendrites and dendritic spines; they also suggest that functional interactions between thalamic axons and subplate neurons could play a role in the establishment of appropriate thalamocortical connections.     

Early postnatal development of cholecystokinin-immunoreactive structures in the visual cortex of the cat

The early postnatal development of cholecystokinin-immunoreactive (CCK-ir) neurons was analyzed in visual areas 17 and 18 of cats aged from postnatal day 0 to adulthood. Neurons were classified mainly by axonal criteria. According to their chronology of appearance neurons are grouped into three neuronal populations. The first population consists of five cell types which appear perinatally in areas 17 and 18. Four of them have axons terminating in layer VI. Neurons with columnar dendritic fields of layers IV and V display a conspicuous dendritic arborization with the long dendrites always arranged parallel to each other. This way they form a vertically oriented dendritic column. The neurons differentiate at around P 2 and are present until the end of the second postnatal week. They disappear possibly by degeneration and cell death. Multipolar neurons of layer VI have long dendrites and axonal domains of up to 800 micron in diameter. Three percent of these neurons send out two axons instead of only one. Neurons differentiate at P 0 and the cell type persists into adulthood. Bitufted to multipolar neurons of layer V constitute a frequent type; 10% of these cells issue two axons. They differentiate at P 2 and the type survives into adulthood. Bitufted to multipolar neurons of layers II/III appear at P 2 and send their axons into layer VI. So, early postnatally an axonal connection from superficial cortical layers to layer VI is established. The cell type persists into adulthood. The fifth cell type of the first population is constituted by the neurons of layer I with intralaminar axons which differentiate at P 2. Although they derive from the early marginal zone, the cell type survives into adulthood. The second population consists of two cell types which appear around the end of the second and during the third postnatal week in areas 17 and 18. Multipolar neurons of layer II have horizontally or obliquely arranged basket axons which, during the second postnatal month, form patches of high fiber and terminal density along the layer I/II border. Neurons with descending main axons issuing horizontal and oblique collaterals of layers II-IV form broad axonal fields. The third population in area 17 is constituted by three cell types: Bitufted neurons with axons descending in form of loose bundles of layers II/III differentiate during the fifth postnatal week. Small basket cells of layers II/III with locally restricted axonal plexuses and somewhat larger basket cells of layer IV appear during the sixth and seventh week.(ABSTRACT TRUNCATED AT 400 WORDS)