Formation of mnemonic neuronal responses to visual paired associates in inferotemporal cortex is impaired by perirhinal and entorhinal lesions.

Functional roles of the cortical backward signal in long-term memory formation were studied in monkeys performing a visual pair-association task. Before the monkeys learned the task, the anterior commissure was transected, disconnecting the anterior temporal cortex of each hemisphere. After training with 12 pairs of pictures, single units were recorded from the inferotemporal cortex of the monkeys as the control. By injecting a grid of ibotenic acid, we unilaterally lesioned the entorhinal and perirhinal cortex, which provides massive direct and indirect backward projections ipsilaterally to the inferotemporal cortex. After the lesion, the monkeys fixated the cue stimulus normally, relearned the preoperatively learned set (set A), and learned a new set (set B) of paired associates. Then, single units were recorded from the same area as for the prelesion control. We found that (i) in spite of the lesion, the sampled neurons responded strongly and selectively to both the set A and set B patterns and (ii) the paired associates elicited significantly correlated responses in the control neurons before the lesion but not in the cells tested after the lesion, either for set A or set B stimuli. We conclude that the ability of inferotemporal neurons to represent association between picture pairs was lost after the lesion of entorhinal and perirhinal cortex, most likely through disruption of backward neural signals to the inferotemporal neurons, while the ability of the neurons to respond to a particular visual stimulus was left intact.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression.

We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.

An anatomical substrate for experience-dependent plasticity of the rat barrel field cortex.

The objective of this study was to examine the influence of sensory experience on the synaptic circuitry of the cortex. For this purpose, the quantitative distribution of the overall and of the gamma-aminobutyric acid (GABA) population of synaptic contacts was investigated in each layer of the somatosensory barrel field cortex of rats which were sensory deprived from birth by continuously removing rows of whiskers. Whereas there were no statistically significant changes in the quantitative distribution of the overall synaptic population, the number and proportion of GABA-immunopositive synaptic contacts were profoundly altered in layer IV of the somatosensory cortex of sensory-deprived animals. These changes were attributable to a specific loss of as many as two-thirds of the GABA contacts targeting dendritic spines. Thus, synaptic contacts made by GABA terminals in cortical layer IV and, in particular, those targeting dendritic spines represent a structural substrate of experience-dependent plasticity. Furthermore, since in this model of cortical plasticity the neuronal receptive-field properties are known to be affected, we propose that the inhibitory control of dendritic spines is essential for the elaboration of these functional properties.

Receptive field structure in the visual cortex: does selective stimulation induce plasticity?

Sensory areas of adult cerebral cortex can reorganize in response to long-term alterations in patterns of afferent signals. This long-term plasticity is thought to play a crucial role in recovery from injury and in some forms of learning. However, the degree to which sensory representations in primary cortical areas depend on short-term (i.e., minute to minute) stimulus variations remains unclear. A traditional view is that each neuron in the mature cortex has a fixed receptive field structure. An alternative view, with fundamentally different implications for understanding cortical function, is that each cell's receptive field is highly malleable, changing according to the recent history of the sensory environment. Consistent with the latter view, it has been reported that selective stimulation of regions surrounding the receptive field induces a dramatic short-term increase in receptive field size for neurons in the visual cortex [Pettet, M. W. & Gilbert, C. D. (1992) Proc. Natl. Acad. Sci. USA 89, 8366-8370]. In contrast, we report here that there is no change in either the size or the internal structure of the receptive field following several minutes of surround stimulation. However, for some cells, overall responsiveness increases. These results suggest that dynamic alterations of receptive field structure do not underlie short-term plasticity in the mature primary visual cortex. However, some degree of short-term adaptability could be mediated by changes in responsiveness.

Object-related activity revealed by functional magnetic resonance imaging in human occipital cortex.

The stages of integration leading from local feature analysis to object recognition were explored in human visual cortex by using the technique of functional magnetic resonance imaging. Here we report evidence for object-related activation. Such activation was located at the lateral-posterior aspect of the occipital lobe, just abutting the posterior aspect of the motion-sensitive area MT/V5, in a region termed the lateral occipital complex (LO). LO showed preferential activation to images of objects, compared to a wide range of texture patterns. This activation was not caused by a global difference in the Fourier spatial frequency content of objects versus texture images, since object images produced enhanced LO activation compared to textures matched in power spectra but randomized in phase. The preferential activation to objects also could not be explained by different patterns of eye movements: similar levels of activation were observed when subjects fixated on the objects and when they scanned the objects with their eyes. Additional manipulations such as spatial frequency filtering and a 4-fold change in visual size did not affect LO activation. These results suggest that the enhanced responses to objects were not a manifestation of low-level visual processing. A striking demonstration that activity in LO is uniquely correlated to object detectability was produced by the "Lincoln" illusion, in which blurring of objects digitized into large blocks paradoxically increases their recognizability. Such blurring led to significant enhancement of LO activation. Despite the preferential activation to objects, LO did not seem to be involved in the final, "semantic," stages of the recognition process. Thus, objects varying widely in their recognizability (e.g., famous faces, common objects, and unfamiliar three-dimensional abstract sculptures) activated it to a similar degree. These results are thus evidence for an intermediate link in the chain of processing stages leading to object recognition in human visual cortex.

Localization of visual stimuli after striate cortex damage in monkeys: parallels with human blindsight.

Blindsight is a phenomenon in which human patients with damage to striate cortex deny any visual sensation in the resultant visual field defect but can nonetheless detect and localize stimuli when persuaded to guess. Although monkeys with striate lesions have also been shown to exhibit some residual vision, it is not yet clear to what extent the residual capacities in monkeys parallel the phenomenon of human blindsight. To clarify this issue, we trained two monkeys with unilateral lesions of striate cortex to make saccadic eye movements to visual targets in both hemifields under two conditions. In the condition analogous to clinical perimetry, they failed to initiate saccades to targets presented in the contralateral hemifield and thus appeared "blind." Only in the condition where the fixation point was turned off simultaneously with the onset of the target--signaling the animal to respond at the appropriate time--were monkeys able to localize targets contralateral to the striate lesion. These results indicate that the conditions under which residual vision is demonstrable are similar for monkeys with striate cortex damage and humans with blindsight.

Illusory contours activate specific regions in human visual cortex: evidence from functional magnetic resonance imaging.

The neural basis for perceptual grouping operations in the human visual system, including the processes which generate illusory contours, is fundamental to understanding human vision. We have employed functional magnetic resonance imaging to investigate these processes noninvasively. Images were acquired on a GE Signa 1.5T scanner equipped for echo planar imaging with an in-plane resolution of 1.5 x 1.5 mm and slice thicknesses of 3.0 or 5.0 mm. Visual stimuli included nonaligned inducers (pacmen) that created no perceptual contours, similar inducers at the corners of a Kanizsa square that created illusory contours, and a real square formed by continuous contours. Multiple contiguous axial slices were acquired during baseline, visual stimulation, and poststimulation periods. Activated regions were identified by a multistage statistical analysis of the activation for each volume element sampled and were compared across conditions. Specific brain regions were activated in extrastriate cortex when the illusory contours were perceived but not during conditions when the illusory contours were absent. These unique regions were found primarily in the right hemisphere for all four subjects and demonstrate that specific brain regions are activated during the kind of perceptual grouping operations involved in illusory contour perception.

Functional activation of the human ventrolateral frontal cortex during mnemonic retrieval of verbal information.

Regional cerebral blood flow was measured with positron emission tomography during the performance of a verbal free recall task, a verbal paired associate task, and tasks that required the production of verbal responses either by speaking or writing. Examination of the differences in regional cerebral blood flow between these conditions demonstrated that the left ventrolateral frontal cortical area 45 is involved in the recall of verbal information from long-term memory, in addition to its contribution to speech. The act of writing activated a network of areas involving posterior parietal cortex and sensorimotor areas but not ventrolateral frontal cortex.

Inhibitory influence of frontal cortex on locus coeruleus neurons.

The functional influence of the frontal cortex (FC) on the noradrenergic nucleus locus coeruleus (LC) was studied in the rat under ketamine anesthesia. The FC was inactivated by local infusion of lidocaine or ice-cold Ringer's solution while recording neuronal activity simultaneously in FC and LC. Lidocaine produced a transient increase in activity in FC, accompanied by a decrease in LC unit and multiunit activity. This was followed by a total inactivation of FC and a sustained increase in firing rate of LC neurons. Subsequent experiments revealed antidromic responses in the FC when stimulation was applied to the LC region. The antidromic responses in FC were found in a population of neurons (about 8%) restricted to the dorsomedial area, FR2. The results indicate that there is a strong inhibitory influence of FC on the tonic activity of LC neurons. The antidromic responses in FC to stimulation of the LC region suggest that this influence is locally mediated, perhaps through interneurons within the nucleus or neighboring the LC.

Parameters affecting conscious versus unconscious visual discrimination with damage to the visual cortex (V1).

When the visual (striate) cortex (V1) is damaged in human subjects, cortical blindness results in the contralateral visual half field. Nevertheless, under some experimental conditions, subjects demonstrate a capacity to make visual discriminations in the blind hemifield (blindsight), even though they have no phenomenal experience of seeing. This capacity must, therefore, be mediated by parallel projections to other brain areas. It is also the case that some subjects have conscious residual vision in response to fast moving stimuli or sudden changes in light flux level presented to the blind hemifield, characterized by a contentless kind of awareness, a feeling of something happening, albeit not normal seeing. The relationship between these two modes of discrimination has never been studied systematically. We examine, in the same experiment, both the unconscious discrimination and the conscious visual awareness of moving stimuli in a subject with unilateral damage to V1. The results demonstrate an excellent capacity to discriminate motion direction and orientation in the absence of acknowledged perceptual awareness. Discrimination of the stimulus parameters for acknowledged awareness apparently follows a different functional relationship with respect to stimulus speed, displacement, and stimulus contrast. As performance in the two modes can be quantitatively matched, the findings suggest that it should be possible to image brain activity and to identify the active areas involved in the same subject performing the same discrimination task, both with and without conscious awareness, and hence to determine whether any structures contribute uniquely to conscious perception.

Lesion-induced reorganization in the brainstem is not completely expressed in somatosensory cortex.

Electrophysiological and neuroanatomical methods were used to determine the extent to which neonatal forelimb removal altered the organization of the cuneate nucleus and representations of the fore- and hindlimbs in the primary somatosensory cortex of adult rats. Neonatal forelimb removal resulted in invasion of the cuneate nucleus by sciatic nerve primary afferents and development of cuneothalamic projection neurons with split receptive fields that included both the hindlimb and forelimb stump. Mapping in the primary somatosensory cortex of the neonatally manipulated adult rats demonstrated abnormalities, but the major change observed in the cuneate nucleus was demonstrable at only a few (5%) cortical recording sites in the remaining stump representation and there were none at all in the hindlimb representation. These results suggest that lesion-induced brainstem reorganization may be functionally suppressed at either the thalamic or cortical level.

Theory of orientation tuning in visual cortex.

The role of intrinsic cortical connections in processing sensory input and in generating behavioral output is poorly understood. We have examined this issue in the context of the tuning of neuronal responses in cortex to the orientation of a visual stimulus. We analytically study a simple network model that incorporates both orientation-selective input from the lateral geniculate nucleus and orientation-specific cortical interactions. Depending on the model parameters, the network exhibits orientation selectivity that originates from within the cortex, by a symmetry-breaking mechanism. In this case, the width of the orientation tuning can be sharp even if the lateral geniculate nucleus inputs are only weakly anisotropic. By using our model, several experimental consequences of this cortical mechanism of orientation tuning are derived. The tuning width is relatively independent of the contrast and angular anisotropy of the visual stimulus. The transient population response to changing of the stimulus orientation exhibits a slow "virtual rotation." Neuronal cross-correlations exhibit long time tails, the sign of which depends on the preferred orientations of the cells and the stimulus orientation.