Developmental divergence of sensory stimulus representation in cortical interneurons.

Vasocative-intestinal-peptide (VIP+) and somatostatin (SST+) interneurons are involved in modulating barrel cortex activity and perception during active whisking. Here we identify a developmental transition point of structural and functional rearrangements onto these interneurons around the start of active sensation at P14. Using in vivo two-photon Ca2+ imaging, we find that before P14, both interneuron types respond stronger to a multi-whisker stimulus, whereas after P14 their responses diverge, with VIP+ cells losing their multi-whisker preference and SST+ neurons enhancing theirs. Additionally, we find that Ca2+ signaling dynamics increase in precision as the cells and network mature. Rabies virus tracings followed by tissue clearing, as well as photostimulation-coupled electrophysiology reveal that SST+ cells receive higher cross-barrel inputs compared to VIP+ neurons at both time points. In addition, whereas prior to P14 both cell types receive direct input from the sensory thalamus, after P14 VIP+ cells show reduced inputs and SST+ cells largely shift to motor-related thalamic nuclei.

Early γ oscillations synchronize developing thalamus and cortex.

During development, formation of topographic maps in sensory cortex requires precise temporal binding in thalamocortical networks. However, the physiological substrate for such synchronization is unknown. We report that early gamma oscillations (EGOs) enable precise spatiotemporal thalamocortical synchronization in the neonatal rat whisker sensory system. Driven by a thalamic gamma oscillator and initially independent of cortical inhibition, EGOs synchronize neurons in a single thalamic barreloid and corresponding cortical barrel and support plasticity at developing thalamocortical synapses. We propose that the multiple replay of sensory input in thalamocortical circuits during EGOs allows thalamic and cortical neurons to be organized into vertical topographic functional units before the development of horizontal binding in adult brain.

Development of structural and functional connectivity in the thalamocortical somatosensory pathway in the wallaby.

Neuronal activity is implicated as a driving force in the development of sensory systems. In order for it to play a developmental role, however, the pathways involved must be capable of transmitting this activity. The relationship between afferent arrival, synapse formation and the onset of chemical neurotransmission has been examined using the advantageous model of a marsupial mammal, the wallaby (Macropus eugenii), to determine at what stage activity has the capacity to influence cortical development. It is known that thalamocortical afferents arrive in the somatosensory cortex on postnatal day (P)15 and that their growth cones reach to the base of the compact cell zone of the cortical plate. However, electronmicroscopy showed that thalamocortical synapses were absent at this stage. Glutamatergic responses were recorded in the cortex following stimulation of the thalamus in slices at this time but only in magnesium-free conditions. The responses were mediated entirely by N-methyl-d-aspartate (NMDA) receptors. From P28, responses could be recorded in normal magnesium and comprised a dominant NMDA-mediated component and a non-NMDA mediated component. At this time thalamocortical synapses were first identified and they were in the cortical plate. By P63 the non-NMDA-mediated component had increased relative to the NMDA-mediated component, and by P70 layer IV began to emerge and contained thalamocortical synapses. By P76 a fast non-NMDA-mediated peak dominated the response. This coincides with the appearance of cortical whisker-related patches and the onset in vivo of responses to peripheral stimulation of the whiskers.

Regulation of thalamocortical patterning and synaptic maturation by NeuroD2.

During cortical development, both activity-dependent and genetically determined mechanisms are required to establish proper neuronal connectivity. While activity-dependent transcription may link the two processes, specific transcription factors that mediate such a process have not been identified. We identified the basic helix-loop-helix (bHLH) transcription factor Neurogenic Differentiation 2 (NeuroD2) in a screen for calcium-regulated transcription factors and report that it is required for the proper development of thalamocortical connections. In neuroD2 null mice, thalamocortical axon terminals fail to segregate in the somatosensory cortex, and the postsynaptic barrel organization is disrupted. Additionally, synaptic transmission is defective at thalamocortical synapses in neuroD2 null mice. Total excitatory synaptic currents are reduced in layer IV in the knockouts, and the relative contribution of AMPA and NMDA receptor-mediated currents to evoked responses is decreased. These observations indicate that NeuroD2 plays a critical role in regulating synaptic maturation and the patterning of thalamocortical connections.

Whisker maps in marsupials: nerve lesions and critical periods.

In the wallaby, whisker-related patterns develop over a protracted period of postnatal maturation in the pouch. Afferents arrive simultaneously in the thalamus and cortex from postnatal day (P) 15. Whisker-related patterns are first seen in the thalamus at P50 and are well formed by P73, before cortical patterns first appear (P75) or are well developed (P85). This study used the slow developmental sequence and accessibility of the pouch young to investigate the effect of nerve lesions before afferent arrival, or at times when thalamic patterns are obvious but cortical patterns not yet formed. The left infraorbital nerve supplying the whiskers was cut at P0-93 and animals were perfused at P112-123. Sections through the thalamus (horizontal plane) and cortex (tangential) were reacted for cytochrome oxidase to visualize whisker-related patterns. Lesions of the nerve at P2-5, before innervation of the thalamus or cortex, resulted in an absence of patterns at both levels. Lesions from P66-77 also disrupted thalamic and cortical patterns, despite the fact that thalamic patterns are normally well established by P73. Lesions from P82-93 resulted in normal thalamic and cortical patterns. Thus, despite the wallaby having clearly separated times for the development of patterns at different levels of the pathway, these results suggest a single critical period for the thalamus and cortex, coincident with the maturation of the cortical pattern. Possible mechanisms underpinning this critical period could include dependence of the thalamic pattern on corticothalamic activity or peripheral signals to allow consolidation of thalamic barreloids.

Effects of prenatal alcohol exposure on the development of the vibrissal somatosensory cortical barrel network.

We have previously shown that the serotonin (5-HT) and its thalamocortical afferents are compromised by prenatal alcohol exposure (PAE). The development of the sensory cortical barrels is regulated by 5-HT-rich thalamocortical afferents. Therefore, it is hypothesized that PAE will deleteriously affect the postnatal development of the cortical barrel formations. On embryonic day (E)7, C57BL/6 mice were grouped into: Alcohol (Alc), Pair-fed (PF), or Chow, and maintained on diet until E18. On postnatal day 7, cortices were stained with 5-HT for thalamocortical fibers, and a NeuN for identification of mature neurons. The area of the posterior medial barrel subfield (PMBSF), was measured as well as the number of NeuN+ neurons within the barrel patches. Though brain weight and brain volume were similar among the three groups, a significant reduction was seen in total area of the PMBSF, and in the average individual barrel area in the Alc group as compared to Chow. Furthermore, the volumes of the B, but not C row barrels were significantly reduced. Barrels were found missing in layer IV, specifically in the posterior aspects of the A, B, and straddler row in the Alc group. Cell counts demonstrated a nearly 50% reduction in NeuN+ neuron number in both rows. This reduction in size of the PMBSF and fewer neurons within these sensory barreloids may underlie a change in the development of the discriminatory sensitivity of the whiskers and serves as an excellent model for the study of a compromised sensory modality following PAE.

Selective serotonin reuptake inhibitor disrupts organization of thalamocortical somatosensory barrels during development.

To further investigate the role of the transiently expressed serotonin (5-HT) transporter (5-HTT) in the development of thalamic fibers projecting to cortical barrels and the potential developmental changes in neuronal circuitry caused by a selective serotonin reuptake inhibitor (SSRI), paroxetine (5 mg/kg, twice daily, s.c.) or saline was administered to rat pups from postnatal day 0 (P0) to P8. Pups were perfused on P8 for 5-HT immunostaining (-im) to confirm the 5-HT uptake blockade, and 5-HTT-im and phospholipase C-beta1 (PLC-beta1)-im to label the thalamic afferents to barrels and barrel cells respectively. Paroxetine treatment completely blocked 5-HT uptake into the thalamocortical fibers as indicated by the negative 5-HT-im in cortical barrel areas. Organization of thalamic afferents to barrels, indicated by 5-HTT-im or PLC-beta1, was altered in paroxetine-treated pups in the following manners: (1) segregation of thalamocortical fibers was partially disrupted and thalamocortical fibers corresponding to anterior snouts and row A mystacial vibrissae were fused; (2) sizes of the unfused thalamocortical fiber patches related to the long caudal vibrissae in rows B, C, D and E were significantly decreased without changes in the brain weights and cortical areas representing these vibrissae; and (3) thalamocortical fibers corresponding to C4 and D4 vibrissae tended to be closer to each other along the arc while the relative positions of thalamocortical fibers related to the rest of the vibrissae were normal. Our study demonstrated that 5-HTT plays an important role in the refinement, but not the formation, of barrel-like clusters of thalamocortical fibers and that the development of neural circuitry in rodent somatosensory cortex was affected by exposure to a SSRI during thalamocortical synaptic formation.

Clorgyline treatment elevates cortical serotonin and temporarily disrupts the vibrissae-related pattern in rat somatosensory cortex.

Manipulation of cortical serotonin (5-HT) levels in perinatal rodents produces significant alterations in the development of the layer IV cortical representation of the mystacial vibrissae. Monoamine oxidase A (MAO(A)) knockout mice have highly elevated cortical 5-HT and completely lack barrels in somatosensory cortex (S-I). The present study was undertaken to determine whether the effects on thalamocortical development seen in MAO(A) knockout mice can be replicated in perinatal rats treated with an MAO(A) inhibitor and, second, to determine whether these effects persist with continued treatment or after discontinuation of the drug. Littermates were injected with either clorgyline (5 mg/kg) or sterile saline five times daily. Clorgyline administration from birth to postnatal day (P) 6, 8, or 10 produced increases of 1,589.4 +/- 53.3%, 1660.2 +/- 43.1% and 1,700.5 +/- 84.5 %, respectively, in cortical 5-HT as compared with controls. Serotonin immunocytochemistry, 1,1;-dioctadecyl-3,3,3", 3;-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamocortical afferents and Nissl and cytochrome oxidase staining of layer IV cellular aggregates demonstrated that clorgyline treatment from P0 to P6 produced a complete absence of any segmentation of vibrissae-related patches in S-I. However, continued treatment until P8 or P10 did not prevent the appearance of these patches. Animals treated with clorgyline from birth to P6 and killed on P8 or P10 had increases of 546.8 +/- 33.2% and 268.8 +/- 6.3% in cortical 5-HT and they had qualitatively normal vibrissae-related patterns in S-I. These results indicate that clorgyline treatment produces a transient disruption of vibrissae-related patterns, despite the continued presence of elevated cortical 5-HT.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

The Notch signalling pathway in hair growth.

The Notch signalling pathway is an important mediator of cell fate selection whose involvement in epidermal appendage formation is now becoming recognised. Hair follicle development and hair formation involve the co-ordinated differentiation of several different cell types in which Notch appears to have a role. We report intricate expression patterns for the Notch-1 receptor and three ligands, Delta-1, Jagged-1 and Jagged-2 in the hair follicle. Notch-1 is expressed in ectodermal-derived cells of the follicle, in the inner cells of the embryonic placode and the follicle bulb, and in the suprabasal cells of the mature outer root sheath. Delta-1 is only expressed during embryonic follicle development and is exclusive to the mesenchymal cells of the pre-papilla located beneath the follicle placode. Expression of Jagged-1 or Jagged-2 overlaps Notch-1 expression at all stages. In mature follicles, Jagged-1 and Jagged-2 are expressed in complementary patterns in the follicle bulb and outer root sheath, Jagged-1 in suprabasal cells and Jagged-2 predominantly in basal cells. In the follicle bulb, Jagged-2 is localised to the inner (basal) bulb cells next to the dermal papilla which do not express Notch-1, whereas Jagged-1 expression in the upper follicle bulb overlaps Notch-1 expression and correlates with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes.