Developmental refinement of visual callosal inputs to ferret area 17.

Corticocortical connections link visual cortical areas in both the ipsilateral and contralateral hemispheres. We studied the postnatal refinement of callosal connections linking multiple cortical areas with ferret area 17 during the period from just before eye opening (4 weeks) to 10 weeks of age. We aimed to determine (1) whether callosal projections from multiple visual cortical areas to area 17 refine with a similar rate and (2) whether the refinement of callosal projections parallels that of intrahemispheric cortical circuits. We injected the bidirectional tracer CTb into area 17, and mapped the areal and laminar distribution of labeled cells in visual areas of the contralateral hemisphere. Like intrahemispheric projections, callosal inputs to area 17 before eye opening are dominated by Suprasylvian area Ssy (with lesser and comparable input from areas 17, 18, 19, and 21), but within 2 weeks of eye opening are jointly dominated by area 18 and Ssy inputs; however, there are fewer labeled cells in the contralateral hemisphere. Unlike intrahemispheric projections, there is no laminar reorganization of callosal inputs; in all visual areas and at all ages studied, the greatest proportion of callosal projections arises from the infragranular layers. Also, unlike intrahemispheric projections, the peak density of callosal cells in each area projecting to area 17 declines more modestly. These results reveal important similarities and differences in the postnatal reorganization of inter- and intrahemispheric projections to area 17.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Visual Corticocortical Inputs to Ferret Area 18.

Visual cortical areas in the adult mammalian brain are linked by a network of interareal feedforward and feedback circuits. We investigated the topography of feedback projections to ferret (Mustela putorius furo) area 18 from extrastriate areas 19, 21, and Ssy. Our objective was to characterize the anatomical organization of the extrastriate feedback pool to area 18. We also wished to determine if feedback projections to area 18 share similar features as feedback projections to area 17. We injected the tracer cholera toxin B subunit (CTb) into area 18 of adult ferrets to visualize the distribution and pattern of retrogradely labeled cells in extrastriate cortex. We find several similarities to the feedback projection to area 17: (i) Multiple visual cortical areas provide feedback to area 18: areas 19, 21, Ssy, and weaker inputs from posterior parietal and lateral temporal visual areas. Within each area a greater proportion of feedback projections arises from the infragranular than from the supragranular layers. (ii) The cortical area immediately rostral to area 18 provides the greatest proportion of total cortical feedback, and has the greatest peak density of cells providing feedback to area 18. (iii) The spacing (peak cell density and nearest neighbor distances) of cells in extrastriate cortex providing feedback to areas 17 and 18 are similar. However, peak density of feedback cells to area 18 is comparable in the supra- and infragranular layers, whereas peak density of feedback cells to area 17 is higher in the infragranular layers. Another prominent difference is that dorsal area 18 receives a cortical input that area 17 does not: from ventral cortex representing the upper visual field; this appears to be roughly 25% of the feedback input to area 18. Lastly, area 17 receives a greater proportion of cortical feedback from area 21 than from Ssy, whereas area 18 receives more feedback from Ssy than from area 21. While the organization of feedback projections from extrastriate cortex to areas 17 and 18 is broadly similar, the main difference in input topography might arise due to differences in visual field representations of the two areas.

Postnatal refinement of interareal feedforward projections in ferret visual cortex.

We studied the postnatal refinement of feedforward (FF) projections from ferret V1 to multiple cortical targets during the period around eye opening. Our goal was to establish (a) whether the developmental refinement of FF projections parallels that of feedback (FB) cortical circuits, and (b) whether FF pathways from V1 to different target areas refine with a similar rate. We injected the tracer CTb into V1 of juvenile ferrets, and visualized the pattern of labeled axon terminals in extrastriate cortex. Bouton density of FF projections to target areas 18, 19, and 21 declined steadily from 4 to 8 weeks postnatal. However, in area Ssy this decline was delayed somewhat, not occurring until after 6 weeks. During this postnatal period, mean interbouton intervals along individual FF axons to all visual areas increased, and we observed a concomitant moderate decrease in axon density in areas 18, 21, and Ssy. These data suggest that FF circuits linking V1 to its main extrastriate targets remodel largely synchronously in the weeks following eye opening, that FF and FB cortical circuits share a broadly similar developmental timecourse, and that postnatal visual experience is critical for the refinement of both FF and FB cortical circuits.

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

Developmental remodeling of corticocortical feedback circuits in ferret visual cortex.

Visual cortical areas in the mammalian brain are linked through a system of interareal feedforward and feedback connections, which presumably underlie different visual functions. We characterized the refinement of feedback projections to primary visual cortex (V1) from multiple sources in juvenile ferrets ranging in age from 4-10 weeks postnatal. We studied whether the refinement of different aspects of feedback circuitry from multiple visual cortical areas proceeds at a similar rate in all areas. We injected the neuronal tracer cholera toxin B (CTb) into V1 and mapped the areal and laminar distribution of retrogradely labeled cells in extrastriate cortex. Around the time of eye opening at 4 weeks postnatal, the retinotopic arrangement of feedback appears essentially adult-like; however, suprasylvian cortex supplies the greatest proportion of feedback, whereas area 18 supplies the greatest proportion in the adult. The density of feedback cells and the ratio of supragranular/infragranular feedback contribution declined in this period at a similar rate in all cortical areas. We also found significant feedback to V1 from layer IV of all extrastriate areas. The regularity of cell spacing, the proportion of feedback arising from layer IV, and the tangential extent of feedback in each area all remained essentially unchanged during this period, except for the infragranular feedback source in area 18, which expanded. Thus, while much of the basic pattern of cortical feedback to V1 is present before eye opening, there is major synchronous reorganization after eye opening, suggesting a crucial role for visual experience in this remodeling process.

Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.

A critical question in brain development is whether different brain circuits mature concurrently or with different timescales. To characterize the anatomical and functional development of different visual cortical areas, one must be able to distinguish these areas. Here, we show that zinc histochemistry, which reveals a subset of glutamatergic processes, can be used to reliably distinguish visual areas in juvenile and adult ferret cerebral cortex, and that the postnatal decline in levels of synaptic zinc follows a broadly similar developmental trajectory in multiple areas of ferret visual cortex. Zinc staining in all areas examined (17, 18, 19, 21, and Suprasylvian) is greater in the 5-week-old than in the adult. Furthermore, there is less laminar variation in zinc staining in the 5-week-old visual cortex than in the adult. Despite differences in staining intensity, areal boundaries can be discerned in the juvenile as in the adult. By 6 weeks of age, we observe a significant decline in visual cortical synaptic zinc; this decline was most pronounced in layer IV of areas 17 and 18, with much less change in higher-order extrastriate areas during the important period in visual cortical development following eye opening. By 10 weeks of age, the laminar pattern of zinc staining in all visual areas is essentially adultlike. The decline in synaptic zinc in the supra- and infragranular layers in all areas proceeds at the same rate, though the decline in layer IV does not. These results suggest that the timecourse of synaptic zinc decline is lamina specific, and further confirm and extend the notion that at least some aspects of cortical maturation follow a similar developmental timecourse in multiple areas. The postnatal decline in synaptic zinc we observe during the second postnatal month begins after eye opening, consistent with evidence that synaptic zinc is regulated by sensory experience.

Early postnatal lesion of the medial dorsal nucleus leads to loss of dendrites and spines in adult prefrontal cortex.

Research suggests that the medial dorsal nucleus (MD) of the thalamus influences pyramidal cell development in the prefrontal cortex (PFC) in an activity-dependent manner. The MD is reciprocally connected to the PFC. Many psychiatric disorders, such as schizophrenia, affect the PFC, and one of the most consistent findings in schizophrenia is a decrease in volume and neuronal number in the MD. Therefore, understanding the role the MD plays in the development of the PFC is important and may help in understanding the progression of psychiatric disorders that have their root in development. Focusing on the interplay between the MD and the PFC, this study examined the hypothesis that the MD plays a role in the dendritic development of pyramidal cells in the PFC. Unilateral electrolytic lesions of the MD in Long-Evans rat pups were made on postnatal day 4 (P4), and the animals developed to P60. We then examined dendritic morphology by examining MAP2 immunostaining and by using Golgi techniques to determine basilar dendrite number and spine density. Additionally, we examined pyramidal cell density in cingulate area 1 (Cg1), prelimbic region, and dorsolateral anterior cortex, which receive afferents from the MD. Thalamic lesions caused a mean MD volume decrease of 12.4% which led to a significant decrease in MAP2 staining in both superficial and deep layers in all 3 cortical areas. The lesions also caused a significant decrease in spine density and in the number of primary and secondary basilar dendrites on superficial and deep layer pyramidal neurons in all 3 regions. No significant difference was observed in pyramidal cell density in any of the regions or layers, but a nonsignificant increase in cell density was observed in 2 regions. Our data are thus consistent with the hypothesis that the MD plays a role in the development of the PFC and, therefore, may be a good model to begin to examine neurodevelopmental disorders such as autism and schizophrenia.

Slow intrinsic rhythm in the koniocellular visual pathway.

Slow rhythmic changes in nerve-cell activity are characteristic of unconscious brain states and also may contribute to waking brain function by coordinating activity between cortical and subcortical structures. Here we show that slow rhythms are exhibited by the koniocellular (K) pathway, one of three visual pathways beginning in the eye and projecting through the lateral geniculate visual relay nucleus to the cerebral cortex. We recorded activity in pairs and ensembles of neurons in the lateral geniculate nucleus of anesthetized marmoset monkeys. We found slow rhythms are common in K cells but are rare in parvocellular and magnocellular cell pairs. The time course of slow K rhythms corresponds to subbeta (<10 Hz) EEG frequencies, and high spike rates in K cells are associated with low power in the theta and delta EEG bands. By contrast, spontaneous activity in the parvocellular and magnocellular pathways is neither synchronized nor strongly linked to EEG state. These observations suggest that parallel visual pathways not only carry different kinds of visual signals but also contribute differentially to brain circuits at the first synapse in the thalamus. Differential contribution of sensory streams to rhythmic brain circuits also raises the possibility that sensory stimuli can be tailored to modify brain rhythms.

Comparison of spatial summation properties of neurons in macaque V1 and V2.

In visual cortex, responses to stimulation of the receptive field (RF) are modulated by simultaneous stimulation of the RF surround. The mechanisms for surround modulation remain unidentified. We previously proposed that in the primary visual cortex (V1), near surround modulation is mediated by geniculocortical and horizontal connections and far surround modulation by interareal feedback connections. To understand spatial integration in the secondary visual cortex (V2) and its underlying circuitry, we have characterized spatial summation in different V2 layers and stripe compartments and compared it to that in V1. We used grating stimuli in circular and annular apertures of different sizes to estimate the extent and sensitivity of RF and surround components in V1 and V2. V2 RFs and surrounds were twice as large as those in V1. As in V1, V2 RFs doubled in size when measured at low contrast. In both V1 and V2, surrounds were about fivefold the size of the RF and the far surround could exceed 12.5 degrees in radius, averaging 5.5 degrees in V1 and 9.2 degrees in V2. The strength of surround suppression was similar in both areas. Thus although differing in spatial scale, the interactions among RF components are similar in V1 and V2, suggesting similar underlying mechanisms. As in V1, the extent of V2 horizontal connections matches that of the RF center, but is much smaller than the largest far surrounds, which likely derive from interareal feedback. In V2, we found no laminar or stripe differences in size and magnitude of surround suppression, suggesting conservation across stripes of the basic circuit for surround modulation.

Retinotopic organization of ferret suprasylvian cortex.

The retinotopic organization of striate and several extrastriate areas of ferret cortex has been established. Here we describe the representation of the visual field on the Suprasylvian visual area (Ssy). This cortical region runs mediolaterally along the posterior bank of the suprasylvian sulcus, and is distinct from adjoining areas in anatomical architecture. The Ssy lies immediately rostral to visual area 21, medial to lateral temporal areas, and lateral to posterior parietal areas. In electrophysiological experiments we made extracellular recordings in adult ferrets. We find that single and multiunit receptive fields range in size from 2 deg x 4 deg to 21 deg x 52 deg. The total visual field representation in Ssy spans over 70 deg in azimuth in the contralateral hemifield (with a small incursion into the ipsilateral hemifield), and from +36 deg to -30 deg in elevation. There are often two representations of the horizontal meridian. Furthermore, the location of the transition from upper to lower fields varies among animals. General features of topography are confirmed in anatomical experiments in which we made tracer injections into different locations in Ssy, and determined the location of retrograde label in area 17. Both isoelevation and isoazimuth lines can span substantial rostrocaudal and mediolateral distances in cortex, sometimes forming closed contours. This topography results in cortical magnifications averaging 0.07 mm/deg in elevation and 0.06 mm/deg in azimuth; however, some contours can run in such a way that it is possible to move a large distance on cortex without moving in the visual field. Because of these irregularities, Ssy contains a coarse representation of the contralateral visual field.

The use of a novel and simple method of revealing neural fibers to show the regression of the lateral geniculate nucleus in the naked mole-rat (Heterocephalus glaber).

The lateral geniculate nucleus (LG) is an important subcortical nucleus in the visual system. It receives primary projections from the retina and relays these to central visual structures. Although there are studies on the retina and visual cortex of animals with regressed vision, little is known about the LG in such animals. The strictly subterranean naked mole-rat (Heterocephalus glaber) has markedly reduced visual acuity with concomitant pronounced changes in the visual cortex. We used a novel method to reveal myelinated neural fibers in a histological study assessing if the LG shows regressive changes commensurate with the level of reliance on vision by this rodent. Myelin detection here relies on significant differences in visible light reflection between neural fibers and the gray matter. Moreover, this simple method does not interfere with further staining for additional analyses. This method reveals that the contribution of the LG to brain volume in the naked mole-rat is less than a third of that of the rat. This shows that the retinogeniculocortical system in the naked mole-rat is considerably smaller than that of rodents that rely heavily on their visual system, but is nevertheless less regressed than that of the extensively studied blind mole-rat; this may facilitate limited responses to visual stimuli.

Feedback connections to ferret striate cortex: direct evidence for visuotopic convergence of feedback inputs.

Interareal feedback connections are a fundamental aspect of cortical architecture, yet many aspects of their organization and functional relevance remain poorly understood. Previous studies have investigated the topography of feedback projections from extrastriate cortex to macaque area 17. We have extended this analysis to the ferret. We made restricted injections of cholera toxin B (CTb) into ferret area 17 and mapped the distribution of retrogradely labeled cells in extrastriate cortex. In addition to extensive label spreading within area 17, we found dense cell label in areas 18, 19, and 21 and the suprasylvian cortex and sparser connections from the lateral temporal and posterior parietal cortex. We made extensive physiological assessments of magnification factors in the extrastriate visual cortex and used these measures to convert the spread of labeled cortex in millimeters into a span in degrees of visual field. We also directly measured the visuotopic extents of receptive fields in the regions containing labeled cells in cases in which we made both CTb injections and physiological recordings in the same animals; we then compared the aggregate receptive field (ARF) of the labeled region in each extrastriate area with that of the injection site. In areas 18, 19, and 21, receptive fields of cells in regions containing labeled neurons overlapped those at the injection site but spanned a greater distance in visual space than the ARF of the injection site. The broad visuotopic extent of feedback connections is consistent with the suggestion that they contribute to response modulation by stimuli beyond the classical receptive field.

A new chamber method for mounting tissue sections.

We describe a method for mounting tissue sections onto slides that is faster and especially useful for those sections too thin or too large to be mounted easily by conventional methods. We have devised a chamber system for mounting tissue sections onto slides under buffer solution. Because of the buoyancy of sections in the buffer solution and the elimination of fluid surface tension, it is easy to move, turn, unfold, and spread even quite large or thin tissue sections. In-solution-mounting in this chamber also greatly reduces the force of the brush tip used to maneuver sections onto the slide, thus resulting in less damage to the tissue sections. This chamber greatly facilitates mounting multiple tissue sections onto a single slide. The new method is applicable to protocols that stain tissue sections either before (e.g. cytochrome oxidase) or after (e.g. cresyl violet) section mounting.

Circuits for local and global signal integration in primary visual cortex.

Contrast-dependent changes in spatial summation and contextual modulation of primary visual cortex (V1) neuron responses to stimulation of their receptive field reveal long-distance integration of visual signals within V1, well beyond the classical receptive field (cRF) of single neurons. To identify the cortical circuits mediating these long-distance computations, we have used a combination of anatomical and physiological recording methods to determine the spatial scale and retinotopic logic of intra-areal V1 horizontal connections and inter-areal feedback connections to V1. We have then compared the spatial scales of these connectional systems to the spatial dimensions of the cRF, spatial summation field (SF), and modulatory surround field of macaque V1 neurons. We find that monosynaptic horizontal connections within area V1 are of an appropriate spatial scale to mediate interactions within the SF of V1 neurons and to underlie contrast-dependent changes in SF size. Contrary to common beliefs, these connections cannot fully account for the dimensions of the surround field. The spatial scale of feedback circuits from extrastriate cortex to V1 is, instead, commensurate with the full spatial range of center-surround interactions. Thus these connections could represent an anatomical substrate for contextual modulation and global-to-local integration of visual signals. Feedback projections connect corresponding and equal-sized regions of the visual field in striate and extrastriate cortices and cover anisotropic parts of visual space, unlike V1 horizontal connections that are isotropic in the macaque. V1 isotropic connectivity demonstrates that anisotropic horizontal connections are not necessary to generate orientation selectivity. Anisotropic feedback connections may play a role in contour completion.

Anatomical origins of the classical receptive field and modulatory surround field of single neurons in macaque visual cortical area V1.

From the analyses of our own and others' anatomical and physiological data for the macaque visual system, we arrive at a conclusion that three pathways can provide the V1 neuron with access to information from the visual field and affect its response. First, direct thalamic input can determine the size of the initial activating RF at high contrast. Second, lateral connections can enlarge the RF at low contrast by pooling information from larger regions of cortex that are otherwise ineffective when high contrast thalamic input is driving the cortical neuron. Thirdly, feedback from extrastriate cortex (possibly together with overlap or interdigitation of coactive lateral connectional fields within V1) can provide a large and stimulus specific surround modulatory field. The stimulus specificity of the interactions between the center and surround fields, may be due to the orderly, matching structure and different scales of intra-areal and feedback projection excitatory pathways. The observed activity changes of single recorded excitatory neurons could be a result of the relative weight of excitation on the excitatory neurons themselves and on local inhibitory interneurons that synapse on them. Inhibitory basket neurons, driven by the local excitatory neurons, could govern local interactions between cortical patches of different tuning properties, resulting in more distant changes in excitatory input in the laterally connected intra-areal neuronal pools.

The spatial extent over which neurons in macaque striate cortex pool visual signals.

We recorded activity of single units in macaque monkey primary visual cortex (V1) to define the retinotopic extent of the visual inputs that drive or modulate V1 neuron responses in parafoveal and peripheral (calcarine) cortex. We used high-contrast drifting grating stimuli to define the extent of the area over which responses summate and the extent of the receptive-field surround. We found responses of most V1 cells to summate over 1 deg, with a suppressive surround typically twice that in diameter, though for some cells (even in parafoveal V1) surrounds exceeded 13 deg in diameter. Surprisingly, we found no significant laminar differences in these dimensions or in the strength of surround suppression. We found that surround suppression in most cells arises from both the ends and sides of the receptive field. Our measures indicate that the strongest modulatory input arises from regions immediately adjacent to the excitatory summation area. These physiological measures suggest that the high-contrast summation field of V1 neurons can be accounted for by the sum of lateral geniculate nucleus (LGN) inputs offered to the local cortical column, with monosynaptic lateral connections within area V1 adding the larger dimensions of the low-contrast summation field and the near surround. Neither of these inputs suffice to explain the largest surrounds, which most likely derive from feedback from extrastriate visual areas.