Rat visual cortical neurones express TrkA NGF receptor

In this study we report the expression of TrkA receptor within the rat visual cortex during postnatal development and in adulthood, using a specific monoclonal antibody which recognizes the extracellular domain of TrkA receptor. TrkA was not detected by immunohistochemistry at postnatal day 13 (P13), i.e. before eye opening. At P22 TrkA was mostly localised in cortical fibre-like processes. At P39 and P90, TrkA-positive neuronal cell bodies in supragranular and infragranular layers were found. Using double immunohistochemistry, labelled cells were identified as intrinsic cholinergic neurones, and as interneurones expressing calbindin and neuropeptide Y. We conclude that TrkA is expressed in visual cortical neurones during postnatal development and in adulthood and that its pattern of expression is developmentally regulated.     

Experience-dependent expression of Nogo-A and Nogo receptor in the developing rat visual cortex

Nogo-A and Nogo receptor (NgR) expression in the visual cortex following a critical developmental period (postnatal days 20-60) has been previously shown. However, little is known regarding Nogo-A and NgR expression between postnatal day 0 and initiation of the critical period. The present study analyzed Nogo-A and NgR expression at four different time points: postnatal day 0 (P0), before critical period (P14), during critical period (P28), and after critical period (P60). Results showed significantly increased Nogo-A mRNA and protein expression levels in the visual cortex following birth, and expression levels remained steady between P28 and P60. NgR mRNA or protein expression was dramatically upregulated with age and peaked at P14 or P28, respectively, and maintained high expression to P60. In addition, Nogo-A and NgR expression was analyzed in each visual cortex layer in normal developing rats and rats with monocular deprivation. Monocular deprivation decreased Nogo-A and NgR mRNA and protein expression in the rat visual cortex, in particular in layers II-III and IV in the visual cortex contralateral to the deprived eye. These findings suggested that Nogo-A and NgR regulated termination of the critical period in experiencedependent visual cortical plasticity.     

Postnatal development of GFAP in mouse visual cortex is not affected by light deprivation

Mammalian visual cortex is immature at birth and develops gradually during defined postnatal temporal windows. In the present work, we studied the maturation of astrocytes in developing mouse visual cortex (VC). The cellular distribution and the level of glial fibrillary acidic protein (GFAP) were analyzed by immunohistochemistry and Western blotting. Experiments were performed at different postnatal ages: postnatal day 12 (P12), before eye opening; P24, corresponding roughly to the peak of the critical period for monocular deprivation, and P60, after the end of the critical period. At P12, GFAP immunoreactivity (IR) was distributed throughout all cortical layers. At P24, there was a prominent localization of GFAP IR in layers I, II, and VI, while cortical layers III, IV, and V contained no longer GFAP IR cells. No differences were found in GFAP IR between P24 and P60. Western blot analysis revealed a reduction of GFAP expression in the VC at P24 with respect to P12 and no significant difference between P60 and P24. These results show that GFAP expression is modulated during early postnatal development. To know whether visual experience influences the maturation pattern of GFAP expression, mice were dark-reared from P12 to P24. Dark rearing did not change the distribution and the expression of GFAP. Our results indicate that maturation of GFAP expression occurs early in postnatal development in mouse VC. In addition, we showed that GFAP development is not affected by visual deprivation.     

Rapid regulation of brain-derived neurotrophic factor mRNA within eye-specific circuits during ocular dominance column formation.

The neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a candidate retrograde signaling molecule for geniculocortical axons during the formation of ocular dominance columns. Here we examined whether neuronal activity can regulate BDNF mRNA in eye-specific circuits in the developing cat visual system. Dark-rearing throughout the critical period for ocular dominance column formation decreases levels of BDNF mRNA within primary visual cortex, whereas short-term (2 d) binocular blockade of retinal activity with tetrodotoxin (TTX) downregulates BDNF mRNA within the lateral geniculate nucleus (LGN) and visual cortical areas. Brief (6 hr to 2 d) monocular TTX blockade during the critical period and also in adulthood causes downregulation in appropriate eye-specific laminae in the LGN and ocular dominance columns within primary visual cortex. Monocular TTX blockade at postnatal day 23 also downregulates BDNF mRNA in a periodic fashion, consistent with recent observations that ocular dominance columns can be detected at these early ages by physiological methods. In contrast, 10 d monocular TTX during the critical period does not cause a lasting decrease in BDNF mRNA expression in columns pertaining to the treated eye, consistent with the nearly complete shift in physiological response properties of cortical neurons in favor of the unmanipulated eye known to result from long-term monocular deprivation. These observations demonstrate that BDNF mRNA levels can provide an accurate "molecular readout" of the activity levels of cortical neurons and are consistent with a highly local action of BDNF in strengthening and maintaining active synapses during ocular dominance column formation.     

Dendritic BDNF synthesis is required for late-phase spine maturation and recovery of cortical responses following sensory deprivation

Sensory experience in early postnatal life shapes neuronal connections in the brain. Here we report that the local synthesis of brain-derived neurotrophic factor (BDNF) in dendrites plays an important role in this process. We found that dendritic spines of layer 2/3 pyramidal neurons of the visual cortex in mutant mice lacking dendritic Bdnf mRNA and thus local BDNF synthesis were normal at 3 weeks of age, but thinner, longer, and more closely spaced (morphological features of immaturity) at 4 months of age than in wild-type (WT) littermates. Layer 2/3 of the visual cortex in these mutant animals also had fewer GABAergic presynaptic terminals at both ages. The overall size and shape of dendritic arbors were, however, similar in mutant and WT mice at both ages. By using optical imaging of intrinsic signals and single-unit recordings, we found that mutant animals failed to recover cortical responsiveness following monocular deprivation (MD) during the critical period, although they displayed normally the competitive loss of responsiveness to an eye briefly deprived of vision. Furthermore, MD still induced a loss of responsiveness to the closed eye in adult mutant mice, but not in adult WT mice. These results indicate that dendritic BDNF synthesis is required for spine pruning, late-phase spine maturation, and recovery of cortical responsiveness following sensory deprivation. They also suggest that maturation of dendritic spines is required for the maintenance of cortical responsiveness following sensory deprivation in adulthood.     

TRKB signalling controls the expression of N-methyl-d-aspartate receptors in the visual cortex

NMDA receptors (NMDARs) are multimeric proteins, the biological and functional characteristics of which depend on differential subunit assembly during postnatal development. In the present paper, we investigated whether the expression of NMDAR subunits NR1, NR2A, NR2B is influenced by neurotrophins in rat visual cortex. We used a soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB-IgG) in order to block TrkB ligands. TrkB-IgG was released through a cannula implanted in the occipital pole and connected to a mini-osmotic pump. TrkB-IgG was continuously released from postnatal day 20-21 (P20-21) to P36-37. In a different group of animals used as controls, osmotic pumps were filled with saline. Different antibodies were used to stain neurons expressing NR1, NR2A and NR2B. We counted the number of neurons stained for NR2A and NR2B subunits and expressed this as percentage with respect to the total number of cresyl-violet stained neurons in each cortical layer. In the visual cortex of TrkB-IgG-treated rats, the percentage of neurons expressing NR2A was significantly increased in all cortical layers. Concerning the NR2B subunit, the percentage of stained neurons was not significantly different between TrkB-IgG-treated and control rats. The staining level for both NR2A and NR2B, but not NR1, was reduced in all cortical layers in TrkB-IgG-treated animals. In agreement with this result, the endogenous levels of NR2A and NR2B subunits were reduced in TrkB-IgG-treated animals as shown by Western blotting. Thus, TrkB signalling controls the cellular expression of NMDAR subunits in visual cortical neurons during postnatal development.     

Differential effects of quinolinic acid lesions on muscarinic acetylcholine receptors in cat visual cortex during postnatal development

Quinolinic acid (QA) lesions of neurons in cat visual cortex were combined with conventional in vitro autoradiographic methods in order to define the cellular locus of the muscarinic acetylcholine receptor (mAChR). Animals of various postnatal ages had QA unilaterally injected into the visual cortex. Four to fourteen days later they were sacrificed and processed for electron microscopy (EM) or in vitro autoradiography. QA lesions at the various postnatal ages were found to eliminate intrinsic cortical neurons and their processes while leaving intact glia, fibers of passage and axon terminals from outside the lesion zone. Autoradiograms of visual cortex labelled with [3H]QNB (which labels M1 and M2 subtypes) showed an age-dependent loss of binding sites, with the greatest decreases occurring after 65 days postnatal. Examined separately, only the M1 mAChRs labelled with [3H]pirenzepine exhibited these age-dependent alterations. The results indicate a differential distribution of the M1 mAChRs during postnatal development. The loss of receptors late in postnatal life following QA suggests a dominantly neuronal locus; the relatively small loss early in postnatal life suggests a locus on other cellular elements.     

Long term depression is expressed during postnatal development in rat visual cortex: a role for visual experience

Long term forms of synaptic plasticity and in particular LTD/LTP are both present in the mammalian visual cortex. However, while LTP is not inducible in adulthood LTD can be elicited in the mature brain, but its developmental pattern is unknown. Aim of this work was to investigate whether LTD is expressed during postnatal development and if it is modulated by visual experience. To investigate these points we have used rat primary visual cortex slices taken at different stages of functional maturation process, i.e., postnatal day 17 (P17), P23 and P30-35. LTD was assessed by measuring the amplitude of extracellular field potentials recorded in cortical layers 2/3 and elicited by low frequency stimulation to the white matter. LTD was expressed at all ages investigated without significant differences between age groups. These data indicate that LTD developmental expression is not temporally related with the period of functional maturation of rat visual cortex. Dark rearing from birth to P23 resulted in a reduction of LTD amplitude while light deprivation from P17 to P30 did not affect LTD expression in comparison to age matched control values. We suggest that light imprinting is essential for a normal LTD expression during postnatal development.     

Postnatal development of neurons and synapses in the visual and motor cortex of rabbits: a quantitative light and electron microscopic study

Introductory to a morphological investigation on the effects of early visual deprivation and on the critical periods in early postnatal life we have studied quantitatively the normal postnatal growth of neurons and synapses in the visual and motor cortex of rabbits. The major results of this analytical study are: (1) rapid decrease in neuron density and a rapid increase in neuronal volume are observed. They are almost completed at postnatal Day 10, i.e., before natural eye opening. The drop in neuron density is caused to a very large extent by an increase in cortical volume and not by a considerable disappearance of neurons; (2) the formation of synaptic contact zones starts at Day 6 to 7 and is most pronounced between Day 10 and Day 21, i.e., after natural eye opening. At Day 27 synaptic density has reached adult levels in the visual cortex and is in excess of the adult level in the motor cortex. In visual area I and in the motor cortex a significant difference in synaptic increase is observed between the left and right hemisphere, resulting in a lower synaptic density in the left counterparts at Day 27 and in adult animals [56,57]. In the visual cortex a small but highly correlated increase in synaptic vesicle density is observed. In the motor cortex no correlated relation between age and vesicle density is observed. In both cortical areas synaptic vesicle density has reached about 70 percent of the adult level at Day 27; and (3) in newborn and young rabbits the motor cortex seems to be more mature than the visual cortex.     

Protein kinase C immunoreactivity in kitten visual cortex is developmentally regulated and input-dependent

Immunocytochemistry with polyclonal antibodies directed against protein kinase C (PKC) was utilized to investigate the development of the kinase in kitten visual cortex neurons. The immunoreaction product was found at postsynaptic sites at all ages studied. However, PKC was localized in presynaptic terminals only during the first few weeks of postnatal life, during the period when the cortex is most susceptible to visual experience. The overall level of PKC immunoreactivity was high at early postnatal ages (up to 6 weeks) and declined afterwards till adulthood. This decline in reactivity was not equal across the cortex and was particularly marked in the middle cortical layers, especially layer IV. The reduction of PKC immunoreactivity in all cortical layers but layer IV was abolished by isolating a portion of cortex from its neuronal inputs early in life. Indirect evidence points to the lateral geniculate nucleus as the source of input that is required for input-dependent maturational changes in the kinase level. The results reported here suggest that the expression of PKC in kitten visual cortex in not only developmentally regulated but is also use-dependent.     

Postnatal development of GFAP, connexin43 and connexin30 in cat visual cortex

In cat visual cortex, neurons acquire progressively mature functional properties during the first postnatal months. The aim of this study was to analyze the development of astrocytes during this period. The patterns of expression of the glial fibrillary acidic protein (GFAP) as well as of two gap junction proteins expressed in astrocytes, connexin43 (Cx43) and connexin30 (Cx30), were investigated by immunohistochemistry and optical density measurements, in visual cortical areas 17 and 18 at four different ages: 2 weeks (postnatal days 12 to 15, P12-15), 1 month (P27-31), 2 months (P60-62) and beyond 1 year. Since visual experience is a key factor for neural development, the patterns of expression of these three proteins were studied both in normally-reared and monocularly deprived animals. Interestingly, the distribution of GFAP, Cx43 and Cx30 was found to change dramatically but independently of visual experience, during postnatal development, even beyond P60. During the first postnatal month, GFAP and Cx43 were mainly localized in the white matter underlying the visual cortical areas 17 and 18. Then, their distributions evolved similarly with a progressive decrease of their density in the white matter associated with an increase in the cortex. Connexin30 expression appeared only from the second postnatal month, strictly in the cortex and with a laminar distribution which was similar to that of Cx43 at the same age. In adults, a specific laminar distribution was observed, that was identical for GFAP, Cx43 and Cx30: their density was higher in layers II/III and V than in the other cortical layers.     

Experience-dependent switch in sign and mechanisms for plasticity in layer 4 of primary visual cortex

Neural circuits are extensively refined by sensory experience during postnatal development. How the maturation of recurrent cortical synapses may contribute to events regulating the postnatal refinement of neocortical microcircuits remains controversial. Here we show that, in the main input layer of rat primary visual cortex, layer 4 (L4), recurrent excitatory synapses are endowed with multiple, developmentally regulated mechanisms for induction and expression of excitatory synaptic plasticity. Maturation of L4 synapses and visual experience lead to a sharp switch in sign and mechanisms for plasticity at recurrent excitatory synapses in L4 at the onset of the critical period for visual cortical plasticity. The state of maturation of excitatory pyramidal neurons allows neurons to engage different mechanisms for plasticity in response to the same induction paradigm. Experience is determinant for the maturation of L4 synapses, as well as for the transition between forms of plasticity and the mechanisms they may engage. These results indicate a tight correlation between the effects of sensory drive and maturation on cortical neurons and provide a new set of cellular mechanisms engaged in the postnatal refinement of cortical circuits.     

Visual activity is required to maintain the phenotype of supragranular NPY neurons in rat area 17

Visual activity governs the functional maturation of the mammalian visual cortex. We report here, that visual experience is required for stabilizing the phenotype of a subset of cortical interneurons. Neurons expressing neuropeptide Y mRNA (NPY neurons) display a transiently higher expression in the early postnatal visual areas 18a and 17 that is followed by a phenotype restriction during the second postnatal month: about 50% of the NPY neurons in supragranular and infragranular layers of area 18a, and in infragranular layers of area 17 gradually stop the NPY expression. In contrast, the expression remains unchanged in supragranular layers of area 17. Dark rearing rats from birth to up to 100 days does neither prevent the developmental onset of NPY mRNA expression, nor does it prevent the phenotype restriction from occurring. In contrast, in dark reared animals NPY neurons in supragranular layers of area 17 now also undergo a phenotype restriction. Returning animals to light after variable periods of darkness results in an upregulation of NPY mRNA expression selectively in neurons in supragranular layers of area 17. These neurons acquire a constitutive expression during the second postnatal month. This suggests that the phenotypic specification of a distinct subset of cortical interneurons is regulated by visual experience which thus influences on the maturation of the neurochemical architecture of area 17.     

The Distribution of Brain-derived Neurotrophic Factor and its Receptor trkB in Parvlbumin-containing Neurons of the Rat Visual Cortex

We analysed the distribution of brain-derived neurotrophic factor (BDNF) and its receptor trkB in the adult rat visual cortex, paying particular attention to a GABAergic neuronal subpopulation—the parvalburnin-positive cells. We found expression of trkB in the cell body and apical dendrite of pyramidal neurons and in the cell body of non-pyramidal neurons. Double labelling experiments revealed extensive colocalization of parvalbumin and trkB immunoreactivity in non-pyramidal neurons. Interestingly, the trkB-positive pyramidal neurons appeared surrounded by parvalbumin-labelled boutons. The use of double immunohistochemistry and in situ hybridization histochemistry showed that parvalbumin-positive neurons express trkB mRNA. BDNF rnRNA was found in several cells. Coexpression of BDNF mRNA and parvalbumin immunoreactivity was extremely rare. These data strongly suggest that BDNF synthesized by cortical neurons acts as a postsynaptically derived factor for parvalbumin-positive neurons in the adult rat visual cortex.     

Development of basal forebrain projections to visual cortex: Dil studies in rat

We performed experiments using retrograde and anterograde labeling with DiI to examine the development of basal forebrain (BFB) projections to the visual cortex in postnatal rats. DiI placed in occipital cortex led to retrograde labeling of BFB neurons as early as postnatal day 0 (P0); labeled cells were found mainly in the diagonal band complex but also in the medial septum, globus pallidus, and substantia innominata. The retrogradely labeled BFB cells displayed remarkably well-developed dendritic arbors, even in younger animals, and showed increases in soma size, dendritic arbors, and dendritic spines over the first 2 postnatal weeks. Dil placements in the diagonal band led to anterogradely labeled axons in cortex. At early ages (P0–P1), labeled axons were largely confined to white matter. With increasing age, greater numbers of labeled axons were seen in the white matter and in deep cortical layers, and labeled axons extended into superficial layers. The leading edge of labeled fibers reached layer V of visual cortex by P2 and layer IV by P4 and were found throughout the cortical layers by P6. Numbers and densities of labeled axons in visual cortex were greater in older animals, at least through P14. The time of ingrowth of labeled BFB axons into visual cortex indicates that these afferents grow into particular cortical layers after those layers have differentiated from the cortical plate. These data indicate that basal forebrain projections arrive in occipital cortex after cortical lamination is well underway and after the entry of primary thalamocortical projections. © 1995 Wiley-Liss, Inc.     

Calcium calmodulin dependent kinase II in cat visual cortex and its development.

A monoclonal antibody against the alpha-subunit of calcium/calmodulin-dependent protein kinase II (CAM-K II) was used to visualize the kinase in developing kitten visual cortex. CAM-K II was first expressed in neurons of the deep cortical layers (V and VI) at postnatal day 1-4 and appeared in the remaining cortical layers within the first 2 weeks. The level of immunoreactivity declined in cells of layer V and upper layer VI at about 30-40 days of age. By postnatal day 90, the most densely labelled neurons were concentrated in cortical layers II, III, lower layer IV and in layer VI. This laminar pattern remained constant into adulthood. EM studies showed that the kinase was found in both pre- and postsynaptic locations. About twice as many immunopositive neurons were found in cortical layers II-IV and VI in young adult cats when geniculate input was removed by an unilateral thalamic lesion performed early in life. These results indicate that expression of CAM-K II is developmentally regulated in visual cortical neurons; the alteration of immunoreactivity after early LGN lesions suggests that the level of the kinase (or its alpha-subunit) is also regulated by cortical input.