Peter H. Mathers

Distinct Subtypes of Somatostatin-Containing Neocortical Interneurons Revealed in Transgenic Mice

GABA-releasing inhibitory interneurons in the cerebral cortex can be classified by their neurochemical content, firing patterns, or axonal targets, to name the most common criteria, but whether classifications using different criteria converge on the same neuronal subtypes, and how many such subtypes exist, is a matter of much current interest and considerable debate. To address these issues, we generated transgenic mice expressing green fluorescent protein (GFP) under control of the GAD67 promoter. In two of these lines, named X94 and X98, GFP expression in the barrel cortex was restricted to subsets of somatostatin-containing (SOM+) GABAergic interneurons, similar to the previously reported “GIN” line (Oliva et al., 2000), but the laminar distributions of GFP-expressing (GFP+) cell bodies in the X94, X98, and GIN lines were distinct and nearly complementary. We compared neurochemical content and axonal distribution patterns of GFP+ neurons among the three lines and analyzed in detail electrophysiological properties in a dataset of 150 neurons recorded in whole-cell, current-clamp mode. By all criteria, there was nearly perfect segregation of X94 and X98 GFP+ neurons, whereas GIN GFP+ neurons exhibited intermediate properties. In the X98 line, GFP expression was found in infragranular, calbindin-containing, layer 1-targeting (“Martinotti”) cells that had a propensity to fire low-threshold calcium spikes, whereas X94 GFP+ cells were stuttering interneurons with quasi fast-spiking properties, residing in and targeting the thalamo-recipient neocortical layers. We conclude that much of the variability previously attributed to neocortical SOM+ interneurons can be accounted for by their natural grouping into distinct subtypes.

Expression of three Rx homeobox genes in embryonic and adult zebrafish.

The paired-class homeobox gene, Rx, is important in eye development. In this study we analyze expression patterns of three zebrafish Rx genes (Zrx1, 2, 3) in embryos and adults. All three genes show dynamic spatiotemporal patterns of expression. Zrx3 is expressed earliest, in the anteriormost region of the neural plate, in regions that give rise to ventral diencephalon and retinae. As development proceeds, Zrx3 expression is reduced in the lateral optic primordia, and is absent in the optic cup, but is retained at the ventral midline of the diencephalon, and is expressed in hypothalamus in the adult. As the neural retina begins to differentiate, Zrx3 is re-expressed in a subset of cells in the inner nuclear layer, presumably bipolar cells, and this expression is retained in the adult. In contrast, Zrx1/2 have a slightly later onset of expression, are initially coincident with Zrx3, but then become complementary, remaining on in the optic primordia but disappearing from the ventral midline of the diencephalon. Zrx1/2 are down-regulated as the retina differentiates, except in the outer nuclear layer where they continue to be expressed at high levels in cone, but not rod, photoreceptors. This is the first transcription factor described that distinguishes between cone and rod photoreceptors.

Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice.

Summary: Rx plays a critical role in eye formation. Targeted elimination of Rx results in embryos that do not develop eyes. In this study, we have investigated the expression of Otx2, Six3, and Pax6 in Rx deficient embryos. We find that these genes show normal activation in the anterior neural plate in Rx−/− embryos, but they are not upregulated in the area of the neural plate that would form the primordium of the optic vesicle. In contrast, in homozygous Small eye embryos that lack Pax6 function, Rx shows normal activation in the anterior neural plate and normal upregulation in the optic vesicle/retinal progenitor cells. This suggests that neither Rx expression nor the formation of retinal progenitor cells is dependent on a functional copy of the Pax6 gene, but that Pax6 expression and the formation of the progenitor cells of the optic cup is dependent on a functional copy of the Rx gene. genesis 28:135–142, 2000.

Synaptogenesis of the Calyx of Held: Rapid Onset of Function and One-to-One Morphological Innervation

Synaptogenesis during early development is thought to follow a canonical program whereby synapses increase rapidly in number and individual axons multiply-innervate nearby targets. Typically, a subset of inputs then out-competes all others through experience-driven processes to establish stable, long-lasting contacts. We investigated the formation of the calyx of Held, probably the largest nerve terminal in the mammalian CNS. Many basic functional and morphological features of calyx growth have not been studied previously, including whether mono-innervation, a hallmark of this system in adult animals, is established early in development. Evoked postsynaptic currents, recorded from neonatal mice between postnatal day 1 (P1) and P4, increased dramatically from −0.14 ± 0.04 nA at P1 to −6.71 ± 0.65 nA at P4 with sharp jumps between P2 and P4. These are the first functional assays of these nascent synapses for ages less than P3. AMPA and NMDA receptor-mediated currents were prominent across this age range. Electron microscopy (EM) revealed a concomitant increase, beginning at P2, in the prevalence of postsynaptic densities (16-fold) and adhering contacts (73-fold) by P4. Therefore, both functional and structural data showed that young calyces could form within 2 d, well before the onset of hearing around P8. Convergence of developing calyces onto postsynaptic targets, indicative of competitive processes that precede mono-innervation, was rare (4 of 29) at P4 as assessed using minimal stimulation electrophysiology protocols. Serial EM sectioning through 19 P4 cells further established the paucity (2 of 19) of convergence. These data indicate that calyces of Held follow a noncanonical program to establish targeted innervation that occurs over a rapid time course and precedes auditory experience.

Expression of a Xenopus Distal-less homeobox gene involved in forebrain and cranio-facial development

Homeobox-containing genes are thought to perform essential functions in the process of pattern formation in vertebrates and invertebrates. They provide cells with positional information critical for normal embryonic development. Since most of the identified homeobox genes in Xenopus seem to provide positional information for the development of the trunk, we have concentrated on genes that may be specifically involved in the formation of the head region. Using a polymerase chain reaction strategy we have searched for Xenopus homeobox-containing genes that might provide positional cues for correct development of the brain. In this paper we report the identification and cloning of a novel gene that by homology appears to be a member of the Distal-less homeobox gene family. We show that its temporal expression patterns in the cement gland, neural crest derived visceral arches, retina and forebrain, while quite diverse, does suggest shared developmental features which may be required for correct craniofacial development and the regionalization of the Xenopus brain. Furthermore, expression of this gene at later stages is primarily restricted to the tadpole forebrain suggesting that the Distal-less gene product continues to play a role after the initial brain patterning is complete.

Maturation of synaptic partners: functional phenotype and synaptic organization tuned in synchrony

Maturation of principal neurons of the medial nucleus of the trapezoid body (MNTB) was assessed in the context of the developmental organization and activity of their presynaptic afferents, which grow rapidly to form calyces of Held and to establish mono‐innervation between postnatal days (P)2 and 4. MNTB neurons and their inputs were studied from embryonic day (E)17, when the nucleus was first discernable, until P14 after the onset of hearing. Using a novel slice preparation containing portions of the cochlea, cochlear nucleus and MNTB, we determined that synaptic inputs form onto MNTB neurons at E17 and stimulation of the cochlear nucleus can evoke action potentials (APs) and Ca2+ signals. We analysed converging inputs onto individual MNTB neurons and found that competition among inputs was resolved quickly, as a single large input, typically larger than 4 nA, emerged from P3–P4. During calyx growth but before hearing onset, MNTB cells acquired their mature, phasic firing property and quantitative real‐time PCR confirmed a coincident increase in low threshold K+ channel mRNA. These events occurred in concert with an increase in somatic surface area and a 7‐fold increase in the current threshold (30 to >200 pA) required to evoke action potentials, as input resistance (Rin) settled from embryonic values greater than 1 GΩ to approximately 200 MΩ. We postulate that the postsynaptic transition from hyperexcitability to decreased excitability during calyx growth could provide a mechanism to establish the mature 1:1 innervation by selecting the winning calyceal input based on synaptic strength. By comparing biophysical maturation of the postsynaptic cell to alterations in presynaptic organization, we propose that maturation of synaptic partners is coordinated by synaptic activity in a process that is likely to generalize to other neural systems.

Molecular Guidance Cues Necessary for Axon Pathfinding from the Ventral Cochlear Nucleus

During development, multiple guidance cues direct the formation of appropriate synaptic connections. Factors that guide developing axons are known for various pathways throughout the mammalian brain; however, signals necessary to establish auditory connections are largely unknown. In the auditory brainstem the neurons whose axons traverse the midline in the ventral acoustic stria (VAS) are primarily located in the ventral cochlear nucleus (VCN) and project bilaterally to the superior olivary complex (SOC). The circumferential trajectory taken by developing VCN axons is similar to that of growing axons of spinal commissural neurons. Therefore, we reasoned that netrin‐DCC and slit‐robo signaling systems function in the guidance of VCN axons. VCN neurons express the transcription factor, mafB, as early as embryonic day (E) 13.5, thereby identifying the embryonic VCN for these studies. VCN axons extend toward the midline as early as E13, with many axons crossing by E14.5. During this time, netrin‐1 and slit‐1 RNAs are expressed at the brainstem midline. Additionally, neurons within the VCN express RNA for DCC, robo‐1, and robo‐2, and axons in the VAS are immunoreactive for DCC. VCN axons do not reach the midline of the brainstem in mice mutant for either the netrin‐1 or DCC gene. VCN axons extend in pups lacking netrin‐1, but most DCC‐mutant samples lack VCN axonal outgrowth. Stereological cell estimates indicate only a modest reduction of VCN neurons in DCC‐mutant mice. Taken together, these data show that a functional netrin‐DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem. J. Comp. Neurol. 504:533–549, 2007.

Rax is a selector gene for mediobasal hypothalamic cell types.

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

Does the brain connect before the periphery can direct? A comparison of three sensory systems in mice.

The development of peripheral to central neural connections within the auditory, visual, and olfactory systems of mice is reviewed to address whether peripheral signaling may play an instructive role during initial synapse formation. For each sensory system, developmental times of histogenesis and the earliest ages of innervation and function are considered for peripheral and selected central relays. For the auditory and visual system, anatomical and functional reports indicate that central connections may form prior to synapse formation in the periphery. However, evidence from the olfactory system suggests that the peripheral olfactory sensory neurons form synaptic connections before more central olfactory connections are established. We find that significant gaps in knowledge exist for embryonic development of these systems in mice and that genetic tools have not yet been systematically directed to address these issues.

Detection of fungi in the sinus mucosa using polymerase chain reaction

OBJECTIVE: To compare the presence of fungi in the sinus mucosa of patients with and without chronic rhinosinusitis. STUDY DESIGN AND SETTING: Prospective observational study using polymerase chain reaction and conventional culture to detect fungi in the sinus mucosa. Middle meatus mucosal samples were collected from 31 patients with chronic rhinosinusitis and 14 control subjects. RESULTS: Fungi were detected in 6.5% of subjects with chronic rhinosinusitis and in none of the control subjects using polymerase chain reaction. Fungi were detected in 29% of subjects with the combination of inhalant allergies, nasal polyposis, and asthma. Fungi were detected in none of the subjects without the combination of these three comorbidities (P = 0.03). CONCLUSION: Polymerase chain reaction assay appears to be able to detect fungi in chronic rhinosinusitis. SIGNIFICANCE: Fungi may not be implicated in the pathogenesis of most chronic rhinosinusitis.