Ferret Visual Cortex Research Articles

In vivo Evidence for Radial Migration of Neurons by Long-Distance Somal Translocation in the Developing Ferret Visual Cortex

During the development of the cerebral cortex, neurons generated in the cortical ventricular zone migrate radially toward the marginal zone. Radially migrating neurons are thought to display 1 of 2 morphologies: cells with a long, pia-contacting, apical process utilized for somal translocation early in development, when the cortex is still relatively thin; or cells with a short leading process, abundant at late stages of corticogenesis when neurons need to travel for longer distances. In large convoluted brains, like those of many primates and carnivores, radially migrating neurons must travel distances up to several millimeters before reaching their final destination, often following curvilinear trajectories. Here we analyze modes and morphologies of radially migrating neurons in convoluted brains by studying the visual cortex of developing ferrets. We provide in vivo and in vitro evidence for the existence of late-born cortical neurons that migrate radially by long-distance somal translocation within a long apical process extended to the cortical plate, in contrast to the early somal translocation observed in rodents. Long-distance translocating neurons in the ferret show a discontinuous rhythm of migration, alternating periods of advance with periods of stall. Furthermore, by combining different labeling methods we find the simultaneous presence in the developing ferret cortex of long-distance translocating neurons and neurons migrating within a short leading process.

Orientation Tuning, But Not Direction Selectivity, Is Invariant to Temporal Frequency in Primary Visual Cortex

The activity of neurons in primary visual cortex is influenced by the orientation, contrast, and temporal frequency of a visual stimulus. This raises the question of how these stimulus properties interact to shape neuronal responses. While past studies have shown that the bandwidth of orientation tuning is invariant to stimulus contrast, the influence of temporal frequency on orientation-tuning bandwidth is unknown. Here, we investigate the influence of temporal frequency on orientation tuning and direction selectivity in area 17 of ferret visual cortex. For both simple cells and complex cells, measures of orientation-tuning bandwidth (half-width at half-maximum response) are approximately 20-25 degrees across a wide range of temporal frequencies. Thus cortical neurons display temporal-frequency invariant orientation tuning. In contrast, direction selectivity is typically reduced, and occasionally reverses, at nonpreferred temporal frequencies. These results show that the mechanisms contributing to the generation of orientation tuning and direction selectivity are differentially affected by the temporal frequency of a visual stimulus and support the notion that stability of orientation tuning is an important aspect of visual processing.

Quantitative morphology and postsynaptic targets of thalamocortical axons in critical period and adult ferret visual cortex

Thalamocortical axons segregate into ocular dominance columns several weeks before the onset of critical period plasticity in ferret visual cortex, a stage characterized by anatomical changes in thalamic input as a consequence of abnormal visual stimulation. In search of possible anatomical correlates of this plasticity, we examined, at electron microscope resolution, the morphology and the synapsing and target selection properties of thalamic axons in ferret visual cortex during and after the critical period. Adult thalamocortical terminals visualized by anterograde tract-tracing display significantly larger cross-section areas than terminals at postnatal day (P) 35, P40, and P49 critical period ages. They are also significantly larger than nonthalamocortical terminals, which attain an adult-like size distribution by P40. The synaptic zones of adult thalamocortical terminals are significantly larger than those of critical period terminals. Perforated and invaginated synapses are encountered frequently on thalamic axons in both adulthood and the P40-49 age group. This result contradicts the view that synaptic perforations and spinules are indicative of a capacity for plasticity. It also suggests that at least some morphological features of thalamic terminals attain maturity on a developmental schedule that is independent of critical period plasticity. Connectivity properties of labeled axons, however, suggest an active role for thalamocortical axons in the critical period. In P40, P49, and adult brains, 23%, 17%, and 9%, respectively, of all thalamocortical synapses contact GABAergic interneurons, suggesting that thalamic input is more strongly involved in driving inhibitory circuits in young ages. Furthermore, thalamocortical axons in P35-49 brains form about 60% more synapses per axon length than in adult brains, suggesting that stabilization of thalamic synapses at the end of the critical period may be accompanied by a reduction of synaptic contacts, as well as a reorganization of postsynaptic circuit selectivity.

VGluT2 immunochemistry identifies thalamocortical terminals in layer 4 of adult and developing visual cortex

A vesicular glutamate transporter, VGluT2, has been suggested to be the transporter utilized in the thalamocortical pathway. We examined the reliability of this marker in identifying and discriminating thalamic terminals in adult and developing ferret visual cortex. We studied brain sections stained for the transporter protein and/or anterogradely filled thalamocortical or intracortical axons, by using light, confocal, and electron microscopy. Under light microscopy, VGluT2 immunoreactivity (ir) in adult animals [past postnatal day (P)90] and in neonatal animals as early as P27 formed a dense band in layer 4 and appeared as scattered puncta in layers 6 and 1. Confocal dual-labeling analyses of P46 and adult striate cortices indicated that VGluT2 was present in thalamocortical axons, suggesting that thalamic projections utilize this transporter during postnatal development as well as adulthood. In contrast, extracellularly filled intracortical axons failed to colocalize with VGluT2-ir, suggesting that no significant terminal population originating in cortex contained VGluT2 in layer 4. Electron microscopic analysis revealed that, in adult layer 4, VGluT2-ir was present in large terminals, forming asymmetric synapses. Similar to anterogradely labeled thalamocortical terminals, VGluT2-ir synaptic terminals were different from their unlabeled counterparts in terms of terminal area (0.6 vs. 0.3 microm), synaptic length (486 vs. 353 nm), and preference for synapsing on spines (77% vs. 59%). Moreover, no significant differences were found between VGluT2-ir and anterogradely labeled thalamocortical terminals. Comparable similarities were also demonstrated at P46. These results indicate that thalamocortical terminals in layer 4 of visual cortex utilize VGluT2 and suggest that this marker can be used to identify thalamic axons specifically in adult and developing animals.

Representing multiple stimulus features in functional maps: Insights from ferret visual cortex

Representing multiple stimulus features in functional maps: Insights from ferret visual cortex

Spatial distribution of inhibitory synaptic connections during development of ferret primary visual cortex

Intracortical inhibition in the primary visual cortex plays an important role in creating properties like orientation and direction selectivity. However, the development of the spatial pattern of inhibitory connections is largely unexplored. This was investigated in the present study. Tangential slices of layers 2/3 of ferret striate cortex were prepared for whole-cell patch clamp recordings, and presynaptic inhibitory inputs to pyramidal neurons were scanned by local photolysis of Nmoc-caged glutamate. Inhibitory synaptic currents (IPSCs) were first detected around postnatal day (P) 17. They originated locally around the recorded cells. Both the number and the total areas supplying the inhibitory inputs increased thereafter and peaked at the time around and shortly after eye opening (P29-37). A refinement period then followed in which the areas providing the majority of inhibitory inputs shrank from 600 microm around the recorded neurons to 200-300 microm in more mature animals (>/=P38). The amplitude of IPSCs increased progressively with increasing age. Long-range inhibitory inputs (>600 microm) were present around eye opening and they often developed into a clustered patchy pattern in more mature animals (>/=P38). In summary, our results show a refinement and clustering in the spatial pattern of inhibitory connections during postnatal development of ferret visual cortex.

Influence of contrast on orientation and temporal frequency tuning in ferret primary visual cortex.

Neurons in primary visual cortex are highly sensitive to the contrast, orientation, and temporal frequency of a visual stimulus. These three stimulus properties can be varied independently of one another, raising the question of how they interact to influence neuronal responses. We recorded from individual neurons in ferret primary visual cortex to determine the influence of stimulus contrast on orientation tuning, temporal-frequency tuning, and latency to visual response. Results show that orientation-tuning bandwidth is not affected by contrast level. Thus neurons in ferret visual cortex display contrast-invariant orientation tuning. Stimulus contrast does, however, influence the structure of orientation-tuning curves as measures of circular variance vary inversely with contrast for both simple and complex cells. This change in circular variance depends, in part, on a contrast-dependent change in the ratio of null to preferred orientation responses. Stimulus contrast also has an influence on the temporal-frequency tuning of cortical neurons. Both simple and complex cells display a contrast-dependent rightward shift in their temporal frequency-tuning curves that results in an increase in the highest temporal frequency needed to produce a half-maximum response (TF(50)). Results show that the degree of the contrast-dependent increase in TF(50) is similar for cortical neurons and neurons in the lateral geniculate nucleus (LGN) and indicate that subcortical mechanisms likely play a major role in establishing the degree of effect displayed by downstream neurons. Finally, results show that LGN and cortical neurons experience a contrast-dependent phase advance in their visual response. This phase advance is most pronounced for cortical neurons indicating a role for both subcortical and cortical mechanisms.

Decline of the Critical Period of Visual Plasticity Is Concurrent with the Reduction of NR2B Subunit of the Synaptic NMDA Receptor in Layer 4

The specific composition of NMDA receptor subunits is thought to underlie the developmental plasticity of the cortex revealed by unbalanced binocular stimulation. However, evidence that NR2 subunits change in correlation with the critical period at locations that are relevant to visual plasticity has been missing. Using preembedding and postembedding immunostaining, as well as electron microscopy, we quantified the volumetric densities of NR1-, NR2A-, and NR2B-containing synapses in layers 4 and 2/3 of the ferret visual cortex at different postnatal ages. Before eye opening, NR2A is encountered infrequently at postsynaptic sites in layer 4, but it increases sharply by postnatal day 34. In the subsequent weeks, postsynaptic NR2A labeling increases gradually in both layers 4 and 2/3 to become the most prevalent subunit in the adult animal. The NR2B subunit is the more prevalent subunit at the onset of the critical period of cortical plasticity. However, it displays different developmental patterns in layers 4 and 2/3. Although no change occurs in synaptic NR2B density in layer 2/3, in layer 4, NR2B maintains its high levels through the peak of the critical period and then becomes significantly reduced by the end of the peak of the critical period. This low level is maintained throughout adulthood. Our results demonstrate a correlation between the loss of NR2B subunits from layer 4 synaptic sites and the decline of the critical period, suggesting that the presence of NR2B subunits at synaptic sites could be a permissive factor regulating the ocular dominance plasticity of the developing cortex.

Coding of Natural Scenes in Primary Visual Cortex

Natural scene coding in ferret visual cortex was investigated using a new technique for multi-site recording of neuronal activity from the cortical surface. Surface recordings accurately reflected radially aligned layer 2/3 activity. At individual sites, evoked activity to natural scenes was weakly correlated with the local image contrast structure falling within the cells' classical receptive field. However, a population code, derived from activity integrated across cortical sites having retinotopically overlapping receptive fields, correlated strongly with the local image contrast structure. Cell responses demonstrated high lifetime sparseness, population sparseness, and high dispersal values, implying efficient neural coding in terms of information processing. These results indicate that while cells at an individual cortical site do not provide a reliable estimate of the local contrast structure in natural scenes, cell activity integrated across distributed cortical sites is closely related to this structure in the form of a sparse and dispersed code.

Receptive Fields and Response Properties of Neurons in Layer 4 of Ferret Visual Cortex

The ferret has become a model animal for studies exploring the development of the visual system. However, little is known about the receptive-field structure and response properties of neurons in the adult visual cortex of the ferret. We performed single-unit recordings from neurons in layer 4 of adult ferret primary visual cortex to determine the receptive-field structure and visual-response properties of individual neurons. In particular, we asked what is the spatiotemporal structure of receptive fields of layer 4 neurons and what is the orientation selectivity of layer 4 neurons? Receptive fields of layer 4 neurons were mapped using a white-noise stimulus; orientation selectivity was determined using drifting, sine-wave gratings. Our results show that most neurons (84%) within layer 4 are simple cells with elongated, spatially segregated, ON and OFF subregions. These neurons are also selective for stimulus orientation; peaks in orientation-tuning curves have, on average, a half-width at half-maximum response of 21.5 +/- 1.2 degrees (mean +/- SD). The remaining neurons in layer 4 (16%) lack orientation selectivity and have center/surround receptive fields. Although the organization of geniculate inputs to layer 4 differs substantially between ferret and cat, our results demonstrate that, like in the cat, most neurons in ferret layer 4 are orientation-selective simple cells.

Spatiotemporal patterns of excitation and inhibition evoked by the horizontal network in layer 2/3 of ferret visual cortex.

The horizontal network in visual cortex layer 2/3 is implicated in numerous psychophysical and physiological properties. To investigate the spatial and temporal distribution of excitation and inhibition evoked by this network, we used voltage-sensitive dyes to image the responses to focal electrical stimulation in tangential slices of ferret visual cortex layer 2/3. The resulting optical patterns included a diffuse zone of activation near the stimulation site and numerous ovoid domains throughout the slice. In contrast to the fixed anatomy of the horizontal connections, substantial shifts in both space and time were evident in the distribution of population-based neuronal activity during stimulus trains. Both of these shifts relied on inhibitory synaptic potentials, suggesting that inhibition driven by horizontal connections sculpts the distribution of activity in this cortical network.

Recruitment of local inhibitory networks by horizontal connections in layer 2/3 of ferret visual cortex.

To investigate how neurons in cortical layer 2/3 integrate horizontal inputs arising from widely distributed sites, we combined intracellular recording and voltage-sensitive dye imaging to visualize the spatiotemporal dynamics of neuronal activity evoked by electrical stimulation of multiple sites in visual cortex. Individual stimuli evoked characteristic patterns of optical activity, while delivering stimuli at multiple sites generated interacting patterns in the regions of overlap. We observed that neurons in overlapping regions received convergent horizontal activation that generated nonlinear responses due to the emergence of large inhibitory potentials. The results indicate that co-activation of multiple sets of horizontal connections recruit strong inhibition from local inhibitory networks, causing marked deviations from simple linear integration.

Reorganization of Exuberant Axonal Arbors Contributes to the Development of Laminar Specificity in Ferret Visual Cortex

Layer-specific cortical axons are believed to develop precisely from the outset without making exuberant branches in incorrect cortical layers. We tested this hypothesis by following the development of axonal arbors of layer 2/3 pyramidal neurons in ferret visual cortex, which in the adult have dense axonal arbors in layers 2/3 and 5 and not in layer 4. We began our studies at earlier ages and sampled at closer intervals than in previous studies. Our studies reveal that between postnatal day (P) 14 and P18 the initial growth of axonal arbors lacks specificity for layers 2/3 and 5 and involves the formation of a small number of branches incorrectly in layer 4. After P18 the number of incorrect axonal branches in layer 4 decreased, whereas there was extensive axonal growth specifically in layers 2/3 and 5. To further study the mechanisms involved in directing the layer-specific growth and elimination of axonal branches, we studied the development of layer 2/3 neurons in slices kept in culture for 5 d. In these studies the initial imprecise growth at P14–18 followed by precise growth after P18 was mimicked <i>in vitro</i>, but the elimination of incorrect axons from layer 4 did not occur. These studies reveal that most axonal arbors grow precisely in the correct layers, but the initial growth involves the formation of a small number of incorrect axonal branches that are later eliminated. Therefore the mechanisms that regulate axonal growth are not as precise initially as later, and mechanisms must exist to allow selective elimination of axon collaterals from incorrect layers.

Reorganization of exuberant axonal arbors contributes to the development of laminar specificity in ferret visual cortex.

Layer-specific cortical axons are believed to develop precisely from the outset without making exuberant branches in incorrect cortical layers. We tested this hypothesis by following the development of axonal arbors of layer 2/3 pyramidal neurons in ferret visual cortex, which in the adult have dense axonal arbors in layers 2/3 and 5 and not in layer 4. We began our studies at earlier ages and sampled at closer intervals than in previous studies. Our studies reveal that between postnatal day (P) 14 and P18 the initial growth of axonal arbors lacks specificity for layers 2/3 and 5 and involves the formation of a small number of branches incorrectly in layer 4. After P18 the number of incorrect axonal branches in layer 4 decreased, whereas there was extensive axonal growth specifically in layers 2/3 and 5. To further study the mechanisms involved in directing the layer-specific growth and elimination of axonal branches, we studied the development of layer 2/3 neurons in slices kept in culture for 5 d. In these studies the initial imprecise growth at P14-18 followed by precise growth after P18 was mimicked in vitro, but the elimination of incorrect axons from layer 4 did not occur. These studies reveal that most axonal arbors grow precisely in the correct layers, but the initial growth involves the formation of a small number of incorrect axonal branches that are later eliminated. Therefore the mechanisms that regulate axonal growth are not as precise initially as later, and mechanisms must exist to allow selective elimination of axon collaterals from incorrect layers.

Relationships of local inhibitory and excitatory circuits to orientation preference maps in ferret visual cortex.

The contribution and precise role of intracortical circuits in generating orientation tuned responses in visual cortical neurons is still controversial. To address this question, the relationship between excitatory and inhibitory synaptic connections and orientation maps in ferret striate cortex was investigated by combining in vivo optical imaging and in vitro scanning laser photostimulation. Excitatory and inhibitory inputs to pyramidal cells originated preferentially from regions with similar orientation preference. Prominent cross-orientation inhibition was not observed, arguing against cross-orientation models of orientation selectivity. The tuning of inhibitory inputs was significantly broader in both layer 2/3 and layer 5/6 pyramidal neurons compared to the tuning of excitatory inputs. Local excitatory inputs were more prominent in the 0-20 degrees tuning difference range between pre- and postsynaptic cells than inhibitory inputs, whereas inhibition dominated in the 20-40 degrees tuning difference range. These differences in tuning of excitatory and inhibitory inputs onto individual cells are consistent with the predictions of recurrent models of orientation selectivity.

The contribution of sensory experience to the maturation of orientation selectivity in ferret visual cortex.

Sensory experience begins when neural circuits in the cerebral cortex are still immature; however, the contribution of experience to cortical maturation remains unclear. In the visual cortex, the selectivity of neurons for oriented stimuli at the time of eye opening is poor and increases dramatically after the onset of visual experience. Here we investigate whether visual experience has a significant role in the maturation of orientation selectivity and underlying cortical circuits using two forms of deprivation: dark rearing, which completely eliminates experience, and binocular lid suture, which alters the pattern of sensory driven activity. Orientation maps were present in dark-reared ferrets, but fully mature levels of tuning were never attained. In contrast, only rudimentary levels of orientation selectivity were observed in lid-sutured ferrets. Despite these differences, horizontal connections in both groups were less extensive and less clustered than normal, suggesting that long-range cortical processing is not essential for the expression of orientation selectivity, but may be needed for the full maturation of tuning. Thus, experience is beneficial or highly detrimental to cortical maturation, depending on the pattern of sensory driven activity.

Development of visual cortical axons: layer-specific effects of extrinsic influences and activity blockade.

During normal cortical development, individual pyramidal neurons form intracortical axonal arbors that are specific for particular cortical layers. Pyramidal neurons within layer 6 are able to develop layer-specific projections in cultured slices of ferret visual cortex, indicating that extrinsic influences, including patterned visual activity, are not required (Dantzker and Callaway [1998] J Neurosci 18:4145-4154). However, when spontaneous activity is blocked in cultures with tetrodotoxin, layer 6 pyramidal neurons fail to preferentially target their axons to layer 4. To determine whether mechanisms that regulate the development of layer 6 pyramidal neuron arbors can be generalized to pyramidal neurons in other layers, we examined the development of layer 5 and layer 2/3 pyramidal neurons in cultured slices of ferret visual cortex prepared on postnatal day 14 or 15. Layer 5 pyramidal neurons developed layer-specific axonal arbors during 5-7 days in vitro. However, unlike layer 6 pyramidal neurons, layer 5 pyramidal neurons formed layer-specific axonal arbors in the presence of tetrodotoxin. In contrast to layer 5 and layer 6 pyramidal neurons, layer 2/3 pyramidal neurons did not form appropriate layer-specific projections during 5-7 days in vitro. Taken together, these data suggest that the development of layer-specific axons is regulated by different mechanisms for neurons in different layers and cannot be categorically classified as either activity-dependent or independent. Instead, the type of pyramidal neuron, the layers targeted, and the type of activity must be considered.

Principal Component Analysis and Blind Separation of Sources for Optical Imaging of Intrinsic Signals

The analysis of data sets from optical imaging of intrinsic signals requires the separation of signals, which accurately reflect stimulated neuronal activity (mapping signal), from signals related to background activity. Here we show that blind separation of sources by extended spatial decorrelation (ESD) is a powerful method for the extraction of the mapping signal from the total recorded signal. ESD is based on the assumptions (i) that each signal component varies smoothly across space and (ii) that every component has zero cross-correlation functions with the other components. In contrast to the standard analysis of optical imaging data, the proposed method (i) is applicable to nonorthogonal stimulus-conditions, (ii) can remove the global signal, blood-vessel patterns, and movement artifacts, (iii) works without ad hoc assumptions about the data structure in the frequency domain, and (iv) provides a confidence measure for the signals (Z score). We first demonstrate on orientation maps from cat and ferret visual cortex, that principal component analysis, which acts as a preprocessing step to ESD, can already remove global signals from image stacks, as long as data stacks for at least two—not necessarily orthogonal—stimulus conditions are available. We then show that the full ESD analysis can further reduce global signal components and—finally—concentrate the mapping signal within a single component both for differential image stacks and for image stacks recorded during presentation of a single stimulus.

Visualizing Neuronal Sturcture by Combining GFP-Transfection With 2-Photon Microscopy In Living Brain Slices

Abstract Research in our lab is aimed at examining how signaling molecules affect the differentiation of neurons in the developing cortex. Most morphological studies rely on single time point images of cells within a population. It is obviously advantageous to examine changes in individual living cells over time. By combining 2-photon imaging with co-transfection of genes for green fluorescent protein (GFP) and neurotrophins in living neurons, we can asses the effects of these on individual living cells. Individual neurons in brain slices from ferret visual cortex were transfected with GFP using particle mediated gene-transfer, or Biolistics. Using the Helios Gene Gun (Bio-Rad), DNA-coated gold particles are blasted into living brain slices. Neurons are transfected when a gold particle lands in the nucleus. Within 24 to 36 hours individual cells are well-filled with GFP allowing visualization of dendrites, spines, and axons.

Unequal representation of cardinal and oblique contours in ferret visual cortex.

We have measured the amount of cortical space activated by differently oriented gratings in 25 adult ferrets by optical imaging of intrinsic signal. On average, 7% more area of the exposed visual cortex was preferentially activated by vertical and horizontal contours than by contours at oblique angles. This anisotropy may reflect the real-world prevalence of contours in the cardinal axes and could explain the greater sensitivity of many animals to vertical and horizontal stimuli.