Ontogeny of the calcium binding protein parvalbumin in the rat nervous system

Summary In the adult rat brain, the calcium-binding protein parvalbumin is preferentially associated with spontaneously fast-firing, metabolically active neurons and coexists with gamma-amino-butyric acid (GABA) in cortical inhibitory interneurons. Whether this is so in developing neurons has not been explored. To this end, we have used parvalbumin immunohistochemistry to study expression of this protein in the rat nervous system during pre- and postnatal life. Our results indicate that parvalbumin first appears at embryonic day 13 in sensory system of the spinal cord, in the vestibular (VIII), the trigeminal (V) and the visuomotor (III, IV VI) systems, and develops rapidly during the following days. In these locations the expression of parvalbumin coincides with the beginning of physiological activity in nerve cells. In the gamma-aminobutyric acid (GABA)-containing interneurons of the cerebral cortex and the hippocampus, as well as in the Purkinje cells of the cerebellum, parvalbumin only appears postnatally. It lags behind the development of GABA-immunoreactivity by 1 to 2 weeks. The beginning of its expression, in the cerebellum at least, coincides with the arrival of excitatory synaptic input and the onset of spontaneous activity. Thus, during the development of the nervous system, the expression of parvalbumin is subordinate to the establishment of physiological activity.Abbreviations 3 oculomotor nucleus - 4 trochlear nucleus - 4n trochlear nerve - 6 abducens nucleus - 12 hypoglossal nucleus - 3n oculomotor nerve - 4V 4th ventricle - 5g trigeminal ganglion - 5n trigeminal nerve - 5mx trigeminal nerve, maxillary branch - 8c1 cochlear ganglion - 8g vestibular ganglion - 8n vestibular nerve - 10n vagal nerve - Amb ambiguus nucleus - CaBP calcium-binding protein - Ce cerebellum - ChP choroid plexus - cl cochlea - CPu caudate putamen - Cu cuneate nucleus - Cx cerebral cortex - df dorsal funiculus spinal cord - dr dorsal root spinal nerve - E15 embryonic day 15 of gestation - ECN external cuneate nucleus - Fr formatio reticularis - GABA gamma-amino-butyric acid - GAD glutamate decarboxylase - gl granular layer cerebellum - Gr gracile nucleus - Hip hippocampus - H heart - inc inferior colliculus - IOK inferior olive, kap cooy medial nucleus - Li liver - LMol lacunosum moleculare layer hippocampus - Lu lung - LV lateral ventricle - LVe(v) lateral vestibular nucleus (ventral) - LVe(d) lateral vestibular nucleus (dorsal) - me5 mesencephalic trigeminal tract - Me5 mesencephalic trigeminal nucleus - Mes mesencephalon - ml molecular layer cerebellum - MVe medial vestibular nucleus - Or oriens layer hippocampus - P 2 postnatal day 2 - Pu Purkinje-cell layer, cerebellum - Py pyramidal cell layer, hippocampus - R red nucleus - Rhom rhombencephalon - Rt reticular thalamic nucleus - sk skin - sn substantia nigra - spgl spinal ganglion - sp5 spinal trigeminal tract - SpVe spinal vestibular nucleus - Sp5I spinal trigeminal nucleus, interpolar - suc superior colliculus - SuVe superior vestibular nucleus - TBS tris-buffered saline - tch tactile hair sinus - ve vestibular epithelium - vb vertebral body - vh ventral horn spinal cord - VL ventrolateral thalamic nucleus - VP ventral pallidum - vr ventral root spinal nerve     

Graded expression of netrin-1 by specific neuronal subtypes in the adult mammalian striatum

Netrins are a family of secreted proteins that function as axon guidance cues during neural development. High levels of netrin-1 expressed by the embryonic ganglionic eminence, the precursor of the adult striatum, direct axons that pioneer the internal capsule. Here we describe netrin-1 expression by neurons distributed throughout the striatum of the adult mouse. Differences were detected in the number and type of neurons expressing netrin-1 in different regions of the striatum. Most neurons in the ventral striatum, including projection neurons and cholinergic interneurons, express netrin-1. In contrast, netrin-1 expression is largely limited to cholinergic interneurons in the dorsal striatum, and the proportion of cholinergic interneurons that express netrin-1 decreases along rostral-caudal and ventral-dorsal axes. Regional differences in expression in the adult striatum suggest that netrin-1 not only influences the development of striatal circuitry but may also participate in the maintenance and plasticity of connections in the adult brain.     

Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons

Sensory information from the periphery is integrated and transduced by excitatory and inhibitory interneurons in the dorsal spinal cord. Recent studies have identified a number of postmitotic factors that control the generation of these sensory interneurons. We show that Gsh1/2 and Ascl1 (Mash1), which are expressed in sensory interneuron progenitors, control the choice between excitatory and inhibitory cell fates in the developing mouse spinal cord. During the early phase of neurogenesis, Gsh1/2 and Ascl1 coordinately regulate the expression of Tlx3, which is a critical postmitotic determinant for dorsal glutamatergic sensory interneurons. However, at later developmental times, Ascl1 controls the expression of Ptf1a in dIL(A) progenitors to promote inhibitory neuron differentiation while at the same time upregulating Notch signaling to ensure the proper generation of dIL(B) excitatory neurons. We propose that this switch in Ascl1 function enables the cogeneration of inhibitory and excitatory sensory interneurons from a common pool of dorsal progenitors.     

Interneurons transiently express the ERG K+ channels during development of mouse spinal networks in vitro

During spinal cord maturation neuronal excitability gradually differentiates to meet different functional demands. Spontaneous activity, appearing early during spinal development, is regulated by the expression pattern of ion channels in individual neurons. While emerging excitability of embryonic motoneurons has been widely investigated, little is known about that of spinal interneurons. Voltage-dependent K+ channels are a heterogeneous class of ion channels that accomplish several functions. Recently voltage-dependent K+ channels encoded by erg subfamily genes (ERG channels) were shown to modulate excitability in immature neurons of mouse and quail. We investigated the expression of ERG channels in immature spinal interneurons, using organotypic embryonic cultures of mouse spinal cord after 1 and 2 weeks of development in vitro. We report here that all the genes of the erg family known so far (erg1a, erg1b, erg2, erg3) are expressed in embryonic spinal cultures. We demonstrate for the first time that three ERG proteins (ERG1A, ERG2 and ERG3) are co-expressed in the same neuronal population, and display a spatio-temporal distribution in the spinal slices. ERG immuno-positive cells, representing mainly GABAergic interneurons, were present in large numbers at early stages of development, while declining later, with a ventral to dorsal gradient. Patch clamp recordings confirmed these data, showing that ventral interneurons expressed functional ERG currents only transiently. Similar expression of the erg genes was observed at comparable ages in vivo. The role of ERG currents in regulating neuronal excitability during the earliest phases of spinal circuitry development will be examined in future studies.     

Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.     

Serotonin regulates voltage-dependent currents in type I(e(A)) and I(i) interneurons of Hermissenda

Serotonin (5-HT) has both direct and modulatory actions on central neurons contributing to behavioral arousal and cellular-synaptic plasticity in diverse species. In Hermissenda, 5-HT produces changes in intrinsic excitability of different types of identified interneurons in the circumesophageal nervous system. Using whole cell patch-clamp techniques we have examined membrane conductance changes produced by 5-HT that contribute to intrinsic excitability in two identified classes of interneurons, types I(i) and I(eA). Whole cell currents were examined before and after 5-HT application to the isolated nervous system. A 4-aminopyridine-sensitive transient outward K(+) current [I(K(A))], a tetraethylammonium-sensitive delayed rectifier K(+) current [I(K(V))], an inward rectifier K(+) current [I(K(IR))], and a hyperpolarization-activated current (I(h)) were characterized. 5-HT decreased the amplitude of I(K(A)) and I(K(V)) in both type I(i) and I(eA) interneurons. However, differences in 5-HT's effects on the activation-inactivation kinetics were observed in different types of interneurons. 5-HT produced a depolarizing shift in the activation curve of I(K(V)) and a hyperpolarizing shift in the inactivation curve of I(K(A)) in type I(i) interneurons. In contrast, 5-HT produced a depolarizing shift in the activation curve and a hyperpolarizing shift in the inactivation curve of both I(K(V)) and I(K(A)) in type I(eA) interneurons. In addition, 5-HT decreased the amplitude of I(K(IR)) in type I(i) interneurons and increased the amplitude of I(h) in type I(eA) interneurons. These results indicate that 5-HT-dependent changes in I(K(A)), I(K(V)), I(K(IR)), and I(h) contribute to multiple mechanisms that synergistically support modulation of increased intrinsic excitability associated with different functional classes of identified type I interneurons.     

Observations on the development of ascending spinal pathways in the clawed toad, Xenopus laevis

Summary The development of ascending spinal pathways has been studied in the clawed toad, Xenopus laevis. From stage 35 (hatching) on, HRP was applied at the spinomedullary border or to the area of the developing dorsal column nucleus, to analyze the development of ascending spinal pathways to the brain stem, and the onset and development of spinal projections to the dorsal column nucleus, respectively. Several populations of spinal neurons with ascending projections at least as far as the spinomedullary border were successively labeled. In early stages ascending spinal projections arise from Rohon-Beard cells and ascending interneuron populations located at the margin of the gray and white matter, i.e., marginal neurons. The ascending interneuron populations could be characterized as dorsolateral commissural and commissural interneurons projecting contralaterally, and as ipsilaterally projecting ascending interneurons and distinguished by Roberts and co-workers. Such a subdivision could be made until about stage 57. Then these ascending and commissural interneuron populations become intermingled with other populations of ascending tract neurons. Rohon-Beard cells could be labeled, more or less shrunken, until stage 55. Around stage 48 (at the time of the appearance of the limb buds) spinal ganglion cells could be labeled from the spinomedullary border and the developing dorsal column nucleus. At stage 48 such ascending primary spinal afferents were found to arise only from non-limbbud-innervating dorsal root ganglia. Gradually also the limb-bud-innervating ganglia give rise to ascending collaterals, so that by stage 53 all spinal ganglia send ascending collaterals to the brain stem. The number of cells of origin of secondary spinal afferents to the brain stem increases during development, and their distribution becomes more extensive. Particularly impressive is a large population of neurons in the dorsal horn projecting ipsilaterally to the dorsal column nucleus. Part of the latter population represents non-primary spinal afferents to the dorsal column nucleus.     

Neuromodulatory role of serotonin in the ferret thalamus

Serotonergic fibers broadly innervate the thalamus and may influence the sleep wake cycle, attention, and other processes through modulation of neurons in this structure. However, the actions of serotonin in the dorsal thalamus have been investigated in detail only in the dorsal lateral geniculate nucleus. In the present study, we examined the action of serotonin in several different regions of the ferret dorsal thalamus, including the associative nuclei, using the in vitro slice preparation and intracellular recording techniques. In nearly all nuclei examined, the predominant action of serotonin was one of hyperpolarization and inhibition of the tonic firing mode. The magnitude of the hyperpolarizing response decreased with age and varied greatly across and somewhat within nuclei maintaining the following relationship (in descending order of magnitude): lateral posterior, lateral dorsal, pulvinar, mediodorsal, center median, anteroventral, central lateral, ventral basal, and medial geniculate. This hyperpolarization is elicited through two mechanisms: one direct and the other via local interneurons. The direct action occurs through an increase in potassium conductance mediated through the 5-HT(1A) receptor. This conclusion is supported by the findings that it persists in the presence of tetrodotoxin and block of GABAergic synaptic transmission, the reversal potential shifts in a Nernstian fashion with changes in extracellular potassium concentration, and the response is antagonized by the 5-HT(1A) antagonist WAY100635 and mimicked by the application of the 5-HT(1A)-selective agonist 8-OH DPAT. The second mechanism by which 5-HT evoked a hyperpolarization was through the activation of local interneurons. In slices in which GABA receptors were not blocked, 5-HT application increased the frequency and amplitude of spontaneous inhibitory postsynaptic potentials (IPSPs) occurring in thalamocortical neurons. Application of 5-HT to physiologically or morphologically identified interneurons evoked a prolonged suprathreshold depolarization. Our results suggest that serotonergic inputs act differentially across the thalamus in a complex manner involving direct and indirect mechanisms. It appears that 5-HT has a greater direct postsynaptic inhibitory influence in the posterior, medial, and intralaminar nuclei than in the primary sensory nuclei.     

Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.     

Primary afferents evoke excitatory amino acid receptor-mediated EPSPs that are modulated by presynaptic GABAB receptors in lamprey.

1. The primary afferent neurons (dorsal cells) are of two types in lamprey, which are fast (touch) and slowly adapting (pressure), respectively. Intracellular stimulation of such sensory neurons evokes mono- and polysynaptic excitatory postsynaptic potentials (EPSPs) in spinobulbar neurons (giant interneurons) and in unidentified interneurons. Paired intracellular recordings between identified sensory cells and spinobulbar neurons made it possible to study the synaptic transmission in detail. It is shown that both touch and pressure primary afferents utilize excitatory amino acid (EAA) transmission and, furthermore, that these effects are subject to a presynaptic GABAB receptor modulation. 2. The monosynaptic mixed electrical and chemical EPSPs in giant interneurons had a mean peak amplitude of 3.2 +/- 1.3 (SD) mV, a time to peak of 4.7 +/- 1.2 ms, and a duration at one-half peak amplitude of 9.4 +/- 3.2 ms. Corresponding results were obtained with dorsal root or dorsal column stimulation. Seventy percent of the fast-adapting dorsal cells of the "touch" type evoked monosynaptic mixed EPSPs in giant interneurons, whereas only 3% of the slowly adapting "pressure" dorsal cells did. 3. The chemical part of the monosynaptic EPSPs evoked in giant interneurons was, in all cases tested, blocked by application of EAA antagonists, like the nonselective antagonist kynurenic acid (KYAC; 2 mM). The selective kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 microM) had a similar effect, whereas the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphono-5-valeric acid (AP-5; 200-400 microM) did not change the EPSP, even in the absence of magnesium ions. 4. The monosynaptic excitatory synaptic transmission was modulated by application of the selective GABAB receptor agonist L-baclofen (5-10 mM local droplet application or 100-1,000 microM bath applied) or by gamma-aminobutyric acid (GABA; 100-1,000 microM), also when GABAA receptor-evoked effects were blocked by bicuculline (10 microM). L-baclofen or GABA in combination with bicuculline did not evoke any effects in the postsynaptic neuron on membrane potential, input resistance, or spike threshold. Therefore the effects of the GABAB receptor activation most likely occurs at the presynaptic afferent level. 5. In conclusion, the monosynaptic excitation from skin mechanoreceptors evoked in spinobulbar neurons is mediated by EAA receptors of the kainate/AMPA type. GABAB receptor activation causes a depression of this EPSP, most likely because of a presynaptic action. GABA interneurons are known to form close appositions on sensory axons in the lamprey.     

Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology

1. As part of a continuing investigation of the organization of the spinal cord of the lamprey, propriospinal interneurons with axons projecting contralaterally and caudally (CC interneurons) were surveyed with intracellular recordings. 2. CC interneurons were identified by recording their axon spikes extracellularly in the spinal cord during intracellular stimulation of the cell body. The axon projections of Cc interneurons were confirmed after intracellular injection and development of horseradish peroxidase. 3. Intracellular stimulation of CC interneurons produced synaptic potentials in myotomal motoneurons, lateral interneurons and other CC interneurons that lay caudally on the opposite side of the spinal cord. Most CC interneurons were inhibitory, but some were excitatory. 4. CC interneurons were divided into three classes on the basis of reticulospinal Müller cell inputs. CC1 interneurons were excited by the ipsilateral Müller cell B1 and the contralateral Mauthner cell. CC1 interneurons were inhibitory. They were excited polysynaptically by ipsilateral sensory dorsal cells and were inhibited by contralateral dorsal cells. They were distinguished morphologically by having no rostral axon branch and no contralateral dendrites. CC1 interneurons were phasically active during fictive swimming with their peak depolarizations preceding those of myotomal motoneurons by about 0.15 cycle. 5. CC2 interneurons were also inhibitory, but they were distinguished from CC1 interneurons by their excitation from the ipsilateral Müller cells B2-4 nd by their thin rostral and thicker caudal axonal branches on the contralateral side of the spinal cord. 6. CC3 interneurons were excitatory, and they were inhibited by the ipsilateral Müller cell I1. CC3 interneurons could have contralateral dendrites and bifurcating axons, and they had lower average axonal conduction velocities than CC1 and CC2 interneurons. 7. Inhibitory CC interneurons may be important for motor coordination in the lamprey. Movements of the lamprey body during reflexes and swimming consist of contraction and relaxation of myotomal muscles on opposite sides of the body. By being coactive with ipsilateral myotomal motoneurons, inhibitory CC interneurons could contribute to the inhibition of contralateral motoneurons during these movements.     

Association interneurons of embryonic rat spinal cord transiently express the cell surface glycoprotein SNAP/TAG-1.

SNAP/TAG-1 is a 135 kDa glycoprotein of the immunoglobulin superfamily that is transiently expressed upon the surfaces of developing axons. In the embryonic rodent spinal cord, this molecule is expressed by motor neurons, dorsal root ganglion cells, and commissural neurons (Yamamoto et al.: J. Neurosci. 6:3576-3594, 1986; Dodd et al.: Neuron 1:105-116, 1988). The commissural cells are a subset of early-forming dorsal horn interneurons whose axons follow a circumferential course in the embryonic spinal cord. The axons of commissural neurons cross the developing ventral commissure to terminate on contralateral synaptic targets, whereas those of the other subset of circumferential cells, the association interneurons, remain on the same side of the spinal cord to form ipsilateral, terminal synaptic fields. The difference between the axonal trajectories of these two subsets of nerve cells raised the question of whether or not association interneurons would also express the SNAP/TAG-1 epitope and, if so, how would this expression be related to that of the commissural cells. Immunocytochemistry for SNAP/TAG-1 and choline acetyltransferase (ChAT) was used to answer these questions. The results indicated that association interneurons expressed SNAP/TAG-1 epitopes and that this expression began later and lasted longer than that of the commissural neurons. Other new findings of this study included the identification of a lateral subgroup of commissural fibers that expressed SNAP/TAG-1 later than their more medially located counterparts, and these lateral fibers were more pronounced in the thoracic spinal cord than at cervical levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinctive pattern of c-fos expression in the feline cervico-lumbar spinal cord after stimulation of vanilloid receptors in dorsal neck muscles

In the present study, c-fos expression in the spinal cord has been used as a marker of neuronal activation induced by capsaicin-sensitive sensory afferents from the dorsal neck muscles in cats (n = 6). The number of Fos-immunoreactive neurons, which were revealed using the avidin-biotin-peroxidase method, was significantly increased in the cervical and lumbar spinal cord. In contrast to the control group (n = 3), 2 h after intramuscular capsaicin injection, c-fos expression was more extensive ipsilaterally to the injected side in the C3-C6 segments, and bilaterally in the L4-L6 segments. Most labeled neurons in the cervical spinal cord were small and giant cells, predominantly located in the middle and lateral parts of lamina I and, additionally, at the neck of the dorsal horn (lamina V), i.e., within the zones of termination of high-threshold muscle afferents. The widespread distribution of labeled cells throughout the cervical cord within the intermediate zone (lamina VII) coincided with the sites of last-order premotor interneurons and cells of origin of long crossed and uncrossed descending propriospinal pathways to the lumbar spinal cord. These findings suggest possible mechanisms for spreading of nociceptive signals between cervical and lumbar regions.     

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta.

Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5-9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells.     

Expression of serotonin 5-HT2C receptors in GABAergic cells of the anterior raphe nuclei

We have used double in situ hybridization to examine the cellular localization of 5-HT2C receptor mRNA in relation to serotonergic and GABAergic neurons in the anterior raphe nuclei of the rat. In the dorsal and median raphe nuclei 5-HT2C receptor mRNA was not detected in serotonergic cells identified as those expressing serotonin (5-HT) transporter mRNA. In contrast, 5-HT2C receptor mRNA was found in most GABAergic cells, recognized by the presence of glutamic acid decarboxylase mRNA. Such 5-HT2C receptor-positive GABAergic neurons were mainly located in the intermediolateral and lateral portions of the dorsal raphe and lateral part of the median raphe. The present data give anatomical support to a previous hypothesis that proposed a negative-feedback loop involving reciprocal connections between GABAergic interneurons bearing 5-HT2A/2C receptors and 5-HT neurons in the dorsal raphe and surrounding areas. According to this model, the excitation of GABAergic interneurons through these 5-HT2C (and also 5-HT2A) receptors would result in the suppression of 5-HT cell firing.