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Depression is a leading cause of disability worldwide, in part because the available treatments are inadequate and do not work for many people. The neurobiology of depression, and the mechanism of action of common antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs), is not well understood. One mechanism thought to underlie the effects of these drugs is upregulation of adult hippocampal neurogenesis. Evidence indicates that vesicular zinc is required for modulation of adult hippocampal neurogenesis, at least under some circumstances. Vesicular zinc refers to zinc that is stored in the synaptic vesicles of certain neurons, including in the hippocampus, and released in response to neuronal activity. It can be eliminated from the brain by deletion of zinc transporter 3 (ZnT3), as is the case in ZnT3 knockout mice. Here, we examined the effects of repeated social defeat stress and subsequent chronic treatment with the SSRI fluoxetine on behaviour and neurogenesis in ZnT3 knockout mice. We hypothesized that fluoxetine treatment would increase neurogenesis and reverse stress-induced behavioural symptoms in wild type, but not ZnT3 knockout, mice. As anticipated, stress induced persistent depression-like effects, including social avoidance and anxiety-like behaviour. Fluoxetine decreased social avoidance, though the effect was not specific to the stressed mice, but did not affect anxiety-like behaviour. Surprisingly, stress increased the survival of neurons born 1 day after the last episode of defeat stress. Fluoxetine treatment also increased cell survival, particularly in wild type mice, though it did not affect proliferation. Our results did not support our hypothesis that vesicular zinc is required for the behavioural benefits of fluoxetine treatment. As to whether vesicular zinc is required for the neurogenic effects of fluoxetine, our results were inconclusive, warranting further investigation into the role of vesicular zinc in adult hippocampal neurogenesis.

Zinc is an important element in all cells of the body, having structural, enzymatic, and regulatory functions. In some neurons, zinc is loaded into synaptic vesicles by zinc transporter 3 (ZnT3) and released into the synaptic cleft, where it can modulate neuronal function. ZnT3 knockout (KO) mice lack ZnT3 and thus lack synaptic zinc. Previous studies have examined the behavioral phenotype of ZnT3 KO mice, mostly using mixed-sex or male-only groups. In the present study we focused specifically on the behavior of female ZnT3 KO mice (2-3 months old). An extensive battery of tests was administered to assess sensorimotor and cognitive behaviours, as well as to examine for a possible schizophrenia-like phenotype. ZnT3 KO mice performed similarly to wild type controls in the majority of tests. However, they were less accurate in the skilled reach task, suggesting impaired skilled motor learning, and faster to descend a vertical pole. ZnT3 KO mice were also slower in the open field and made fewer chamber entries in the social preference test, suggesting decreased exploratory locomotion. No differences were observed in the Morris water task or fear conditioning test. This is the first study to show a behavioural phenotype specifically for female ZnT3 KO mice. Comparing our results to previous studies, it appears that there may be sex-specific effects of eliminating ZnT3. Female ZnT3 KO mice exhibit abnormalities in locomotion and at skilled motor learning, but we were unable to detect spatial or fear learning deficits previously described in male ZnT3 KO mice.

Zinc-containing terminals are found throughout the neocortex, concentrated predominantly in layers II/III, V, and VI. Synaptic zinc is a potent neurotransmitter/modulator and, therefore, may mediate inter- or intra-cortical integration of sensory information. We have previously shown that levels of synaptic zinc are rapidly modulated in somatosensory (barrel) cortex, in an experience- and activity-dependent manner. Zinc transporter 3 (ZnT3) knockout (KO) mice lack synaptic zinc and provide us with a good model to examine the contribution of synaptic zinc to barrel cortex-dependent behavior. In the present study, we show that ZnT3 KO mice display a marked decrease in acuity for whisker-dependent texture discrimination. ZnT3 KO mice were not able to discriminate between textures having an average particle diameter less than 300 μm while control mice were able to discriminate between textures having particle diameters separated by as little as 25 μm. This loss of texture discrimination acuity in ZnT3 KO mice was whisker-dependent and was observed in young (2 months-of-age) and older mice (12 months-of-age). These results show that zincergic signaling is necessary for the normal integration of somatosensory information.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.

Zinc transporter 3 (ZnT3) is the sole mechanism responsible for concentrating zinc ions within synaptic vesicles in a subset of the brain's glutamatergic neurons. This vesicular zinc can then be released into the synaptic cleft in an activity-dependent fashion, where it can exert many signaling functions. This review provides a comprehensive discussion of the localization and function of ZnT3 and vesicular zinc in the central nervous system. We begin by reviewing the fundamentals of zinc homeostasis and transport, and the discovery of ZnT3. We then focus on four main topics. I) The anatomy of the zincergic system, including its development and its modulation through experience-dependent plasticity. II) The role of zinc in intracellular signaling, with a focus on how zinc affects neurotransmitter receptors and synaptic plasticity. III) The behavioural characterization of the ZnT3 KO mouse, which lacks ZnT3 and, therefore, vesicular zinc. IV) The roles of ZnT3 and vesicular zinc in health and disease.

Expansion of motor maps occurs in both clinical populations with epilepsy and in experimental models of epilepsy when the frontal lobes are involved. We have previously shown that the forelimb area of the motor cortex undergoes extensive enlargement after seizures, although the extent to which many movement representation areas are altered is not clear. Here we hypothesize that movement representations in addition to the forelimb area will be enlarged after cortical seizures. To test our hypotheses, Long Evans Hooded rats received 20 sessions of callosal (or sham) kindling, and then were subjected to intracortical microstimulation to map several movement representations including the jaw, neck, forelimb, hindlimb, trunk and tail. We found significantly larger total map areas of several movement representations, including movements that could be evoked more posterior than they are in control rats. We also show the presence of more multiple movement sites and lower movement thresholds in kindled rats, suggesting that movements not only overlap and share cortical territory after seizures, but become present in formerly non-responsive sites as they become detectable with our intracortical microstimulation methodology. In summary, several motor map areas become larger after seizures, which may contribute to the interictal motor disturbances that have been documented in patients with epilepsy.

Increasing evidence suggests that synaptic zinc, found within the axon terminals of a subset of glutamatergic neurons in the cerebral cortex, is intricately involved in cortical plasticity. Using the vibrissae/barrel cortex model of cortical plasticity, we have previously shown manipulations of sensory input leads to rapid changes in synaptic zinc levels within the corresponding regions of the somatotopic map in the cortex. Here, using electron microscopy, we show how some of these changes are mediated at the synaptic level. We found that the density of zincergic synapses increased significantly in layers II/III, IV, and V. In layers IV and V, this change occurred in the absence of a significant increase in excitatory synapse density, which seems to indicate that excitatory synapses, which previously did not contain synaptic zinc, begin to newly house zinc within its synaptic vesicles. Our results show that excitatory neurons can dynamically change the phenotype of the vesicular content of their synapses in response to changes in sensory input. Given the range of modulatory effects zinc can have on neurotransmission, such a change in the complement of vesicular contents presumably allow these neurons to utilize synaptic zinc to facilitate plasticity. Thus, our results further support the role of zinc as an active participant in the processes contributing to experience-dependent cortical plasticity.

Alzheimer's disease (AD) is characterized by distinct behavioral and cognitive deficits that differ from those observed in normal aging. Transgenic models of AD are a promising tool in understanding the underlying mechanisms and cause of disease. The triple-transgenic mouse model of AD (3xTg-AD) is the only model to exhibit both Abeta and tau pathology that is characteristic of the human form. The present study characterized the performance of 3xTg-AD mice on several tasks measuring behavioral and cognitive ability. Aged 3xTg-AD females exhibited a higher level of fear and anxiety demonstrated by increased restlessness, startle responses, and freezing behaviors. No differences were observed in muscle strength and visuo-motor coordination. Understanding the behavioral manifestations that occur in this model of AD may aid in the early diagnosis and appropriate treatment of AD symptomology.

In the mammalian cerebral cortex, zinc is an important modulator of synaptic transmission and conversely, plasticity. Zinc is also involved, in a sex-dependent manner, in the pathogenesis of Alzheimer's disease (AD), where substantial declines in plasticity may occur. To examine this relationship further, the regulation of vesicular zinc was examined after the induction of cortical plasticity through vibrissae plucking in male and female C57Bl/6 and 3xTg-AD mice at various age points. Female C57Bl/6 mice were found to have an elevated response compared to male C57Bl/6 mice through mid-adult ages, a sex-difference likely mediated by the differential regulation of vesicular zinc by the sex hormones. Male 3xTg-AD mice had a significantly greater zincergic response compared to C57Bl/6 mice, which is likely indicative of a compensatory mechanism utilized by the male 3xTg-AD mice to combat the decline in plasticity associated with the AD state. These results exemplify how the regulation of vesicular zinc may be a significant component in the progression of AD, especially regarding the sex-dependent element.

Despite the plethora of reports that demonstrate plasticity in the mammalian cerebral cortex, the characterization of the cellular mechanisms that mediate it is sparse. Here, we show that the magnitude of the experience-dependent regulation of vesicular zinc is significantly increased through enriched-environment housing. Mice were reared either in a deprived environment and subsequently housed in deprived, minimally enriched, or enriched conditions after the removal of the c-row of vibrissae or reared in an enriched environment before and after vibrissae removal. Levels of vesicular zinc were assessed in deprived and nondeprived barrels 6 h to 14 d after vibrissae removal. We found that housing in enriched environmental conditions resulted in a greater change in vesicular zinc levels than did other housing conditions; however, this effect was dependent on both the magnitude and duration of enrichment. Our data indicate that enriched-environment housing has profound effects on the regulation of vesicular zinc that occurs concurrently with experience-dependent plasticity, suggesting a role for zinc in the multitude of cortical modifications associated with enriched environments.

Zinc is released from a subset of cerebral cortical neurons whereupon it exerts a powerful modulatory influence on excitatory and inhibitory neurotransmission. A number of studies have suggested that alterations in the regulation of zinc may contribute to the genesis of epilepsy. Here, we tested this hypothesis by examining the distribution of zinc-containing axon terminals in rats selectively bred for an innate susceptibility (FAST) or resistance (SLOW) to the development of kindling-induced seizures. Zinc was stained histochemically and levels of staining were quantitatively assessed. We found that the levels of synaptic zinc were significantly lower in the SLOW rats throughout the telencephalon. This relative reduction was most pronounced in limbic cortices where levels were less than 30% of FAST rats. These results suggest that innate differences in the homeostatic regulation of synaptic zinc, particularly in limbic cortices, may underlie differences in epileptogenicity.

Injury of the brain is a leading cause of long-term disability. Recent evidence indicates that the selective serotonin reuptake inhibitor drug fluoxetine may be beneficial when administered following brain injury. However, its potential to promote recovery and the mechanisms by which it might do so require further characterization. In the present experiment, fluoxetine was administered to mice for 4 weeks following injury of medial frontal cortex (MFC). MFC injury altered behavior, reducing locomotion, decreasing swim speed in the Morris water task, and decreasing anxiety-like behavior in the elevated plus maze. Fluoxetine treatment did not affect these behavioral alterations, but it did increase the social dominance of the injured mice, as assessed by the tube test. Fluoxetine treatment also hastened learning of a T-maze position discrimination task, independently of lesion condition. Anatomically, fluoxetine failed to decrease lesion size, increase the survival of cells born 1-week post injury in the hippocampal dentate gyrus, or reverse the reduction in spine density in layer II/III pyramidal neurons in cingulate cortex caused by the lesions. Fluoxetine did, however, increase the dendritic arborization of these cells, which was reduced in the mice with lesions. Thus, while not all the effects of MFC injury were ameliorated, the behavioral outcome of mice with MFC injuries was improved, and one of the neuroanatomical sequelae of the lesions counteracted, by chronic fluoxetine, further contributing to the evidence that fluoxetine could be a useful treatment following brain injury.

Intracellular recordings were made in the dorsal hippocampal formation of urethane-anesthetized rats as the local field activity spontaneously cycled between a synchronous condition termed theta and an asynchronous condition termed LIA. All cells reported in this study were labeled with Neurobiotin and classified as theta-related or non-theta-related according to the system of Colom and Bland [Colom, L.V., Bland, B.H., 1987. State-dependent spike train dynamics of hippocampal formation neurons: evidence for theta-ON and theta-OFF cells. Brain Res. 422; 277-286]. The findings are the first demonstration that hippocampal pyramidal cells are functionally heterogeneous in relation to the generation of theta-band oscillation and synchrony. In field CA1 pyramidal cells formed theta-related subsets of phasic theta-ON cells and tonic theta-ON cells and non-theta-related subsets of simple spike discharging cells, complex spike discharging cells and "silent" cells. Similar findings were evident for CA3 pyramidal cells.

Sensory cortices have multiple and distinct functional maps that systematically represent environmental information. Development of these maps is precisely controlled by a number of intrinsic and extrinsic factors. Cortical cholinergic regulation is a crucial factor for normal cortical morphogenesis. In this study, we test the role of the M1 muscarinic acetylcholine receptor, the main muscarinic receptor subtype in the neocortex in the development of tonotopic maps in the auditory cortex. Mice lacking M1 receptors have normal hearing sensitivity but exhibit disrupted tonotopic organization and frequency tuning in the auditory cortex. In contrast, tonotopic organization and frequency tuning remain normal in the auditory midbrain. In addition, cortical layer IV neurons of M1 mutants exhibit significantly shorter or sparser dendrites compared to neurons of wildtype mice. In summary, our data suggest that the M1 receptor appears to be critical for the refinement or normal maturation of cortical tonotopy that is guided by thalamocortical inputs during early development.

The cortical representation of the body surface is not fixed, but rather, is continuously modified by ongoing changes in sensory experience. Although the cellular and molecular mechanisms that subserve these changes are uncertain, increasing evidence suggests that synaptically-released zinc may play a role. Zinc is released from a subset of glutamatergic neurons and can modulate postsynaptic excitability by regulating the activation of glutamate and GABA receptor-gated ion channels. Previously, we have shown that whisker plucking, a manipulation commonly used to induce cortical map plasticity, results in a rapid and robust increase in staining levels for synaptic zinc in deprived regions of the barrel cortex. In the present study, we examined the effect of increased whisker activity, analogous to what may happen during tactile learning or exploratory behavior in a natural setting, on synaptic zinc levels in the adult barrel cortex. Our results indicate that stimulation of whiskers caused a selective decrease in zinc levels within layer 4 of the barrel hollow corresponding to the stimulated whisker. Quantitatively, levels of staining were significantly reduced at 3 h, and showed even greater reductions following 12 and 24 h of stimulation. However, these changes were not long-lasting, as levels of staining in the stimulated barrel returned to control values within 24 h after stimulation had ceased. These data indicate that zincergic circuits are highly sensitive to ongoing changes in sensory experience and may participate in moment-to-moment changes in the functional connectivity of the cerebral cortex.

Degeneration of dopaminergic neurons in the substantia nigra is associated with one of the most prominent human neurological disorders, Parkinson's disease. It is therefore of high interest to identify molecules with trophic effects on this neuronal population. We show here that the neuregulin receptor ErbB4 is differentially expressed in mesencephalic dopaminergic neurons, found in the substantia nigra and in a subregion of the ventral tegmentum but not in the retrorubral field. Early developmental onset and continued expression of ErbB4 into the adult and the presence of two high affinity ligands, neuregulin-1 and betacellulin, in the basal ganglia, suggested that these molecules might participate in the differentiation and/or maintenance of the nigrostriatal system. In order to address this hypothesis, we used a loxP flanked ErbB4 allele in combination with a nestin-Cre transgene and generated brain-specific ErbB4 null mice. These mutant animals survived into adulthood. The distribution of dopaminergic cell bodies in the midbrain, the expression of numerous genes specific to mesencephalic dopaminergic neurons, and the axonal projection to the basal ganglia all appeared normal. Finally, an assessment of their motor function revealed no behavioral deficits. The apparent lack of any mutant phenotype suggests the presence of a strong compensatory mechanism.

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.

Zinc plays an important role in synaptic signaling in the mammalian cerebral cortex. Zinc is sequestered into presynaptic vesicles of subpopulations of glutamatergic neurons and is released by depolarization, in a calcium-dependent manner. As the majority of mechanisms that have been suggested to participate in experience-dependent alterations in synaptic strength in the cerebral cortex implicate signaling by glutamate, it stands to reason that zincergic signaling might also be crucial. Here we show that synaptic zinc is rapidly and dynamically modulated in relation to alterations in sensory input and that this response is highly age-dependent. Juvenile, adult, and aged mice were subjected to whisker removal and levels of staining for synaptic zinc in deprived and non-deprived cortical barrels were quantitatively assessed at post-deprivation times ranging from 3 h to 21 days. In the first 12 h, zinc levels increased slightly, but significantly, in all groups. At later time points, zinc levels increased robustly (23%) in the youngest group by 24 h and remained elevated through 7 days. By contrast, deprivation-induced changes in zinc staining in aged animals, achieved their maximal levels at 12 h (approximately 10%) and steadily declined thereafter. Adult animals revealed a biphasic, intermediate change with time. In all age groups, levels of zinc staining returned to baseline by 21 days after whisker plucking. However, only in juvenile and adult mice did we observe that the level of zinc staining in deprived barrel hollows, was correlated with the length of whiskers as they regrew. Our data suggest that alterations in the regulation of synaptic zinc may be involved with decrements of synaptic plasticity that accompany senescence.

In this paper we emphasize three important aspects of intracellular recording with sharp electrodes and subsequent cell labeling with neurobiotin, utilizing the acute in vivo rat preparation: first, preparing access to the hippocampal formation and overcoming the problems presented by the sagittal sinus in gaining access to the septum for recording; secondly, the mechanical stabilization of the in vivo preparation in order to reduce brain movements due to respiration and heart beat; and thirdly, fabrication and filling of sharp glass recording electrodes which can be used for successful intracellular recording and labeling. We present examples of such successful electrophysiogical recording and labeling of hippocampal and septal neurons.

Intracellular recordings of cells, classified according to the criteria of Colom and Bland as phasic theta-ON or phasic theta-OFF cells, were carried out in the dorsal region of the hippocampal formation in urethan-anesthetized rats. Cells were studied during two spontaneously occurring hippocampal field conditions, asynchrony, termed large-amplitude irregular activity, and synchrony, termed theta. During the spontaneous cycling between these two field states, the effect of four levels of intracellular depolarizing and hyperpolarizing constant current injections on the amplitude and phase of membrane potential oscillations (MPOs) and the rate and pattern of cell discharges was assessed. Labeled CA1 pyramidal cells and bistratified cells met the criteria for classification as phasic theta-ON cells and labeled CA1 pyramidal layer basket cells, mossy hilar cells, and granule cells met the criteria for classification as phasic theta-OFF cells. MPOs were recorded in CA1 pyramidal cells, CA1 layer basket cells, mossy interneurons, and granule cells only during theta field activity, their onset in theta-ON cells signaled by a depolarizing shift of 5-10 mV and in theta-OFF cells by a hyperpolarizing shift of 5-10 mV, in membrane potential. The effect of current injections in phasic theta-ON and theta-OFF cells during the theta field condition revealed that MPO amplitude was voltage dependent and frequency was voltage independent. There were no phase changes observed in phasic theta-ON cells during current injections; however, amplitude analysis revealed an inverted U-shaped curve asymmetrically distributed around the average value of the membrane potential occurring during the spontaneous theta (no current) control condition. The occurrence and rate of rhythmical cell discharges in CA1 pyramidal phasic theta-ON cells during the theta condition was precisely controlled within a critical range of membrane potential values from approximately -57 to -68 mV, corresponding to a range of MPO amplitudes of approximately 4-7 mV. Outside the critical range, rhythmic cell discharges were abolished. Membrane potential oscillations in CA1 pyramidal layer basket cells underwent an approximate 180 degrees phase reversal when the membrane potential was depolarized above -65 mV. The occurrence and rate of rhythmic cell discharges in CA1 pyramidal layer basket cell phasic theta-OFF cells during the theta condition was precisely controlled within a critical range of membrane potential values from approximately -62 to -60 mV, corresponding to a range of MPO amplitudes of approximately 7-7.5 mV. Outside the critical range, cell discharges were absent or occurred singly.

Electrophysiological studies have established that the adult cerebral cortex undergoes immediate functional reorganizations after perturbations of the sensory periphery. These activity-dependent modifications are thought to be mediated via the rapid regulation of the synaptic strength of existing connections. Recent studies have implicated synaptic zinc as contributing to activity-dependent mechanisms of cortical plasticity, such as long-term potentiation and long-term depression, by virtue of its potent ability to modulate glutamatergic neurotransmission. To investigate the role of synaptic zinc in cortical plasticity, we examined changes in the barrel-specific distribution of zinc in axon terminals innervating the primary somatosensory cortex of adult mice at different time points after whisker plucking. In layer IV of normal adult mice, zinc staining in the barrel field was characterized by intense staining in inter-barrel septae and low levels of staining in barrel hollows. Within 3 hr, and up to 1 week after the removal of a row of whiskers, zinc staining increased significantly in barrel hollows corresponding to the plucked whiskers. With longer survival times, levels of zinc staining gradually declined in deprived barrel hollows, returning to normal levels by 2–3 weeks after whisker removal. Increased levels of zinc staining in deprived barrel hollows were highly, negatively correlated with the length of whiskers as they regrew. These results indicate that levels of synaptic zinc in the neocortex are rapidly regulated by changes in sensory experience and suggest that zinc may participate in the plastic changes that normally occur in the cortex on a moment-to-moment basis.

The subgranular zone of the hippocampal formation gives rise to new neurons that populate the dentate gyrus throughout life. Cells in the hippocampus exhibit rhythmic clock gene expression and the circadian clock is known to regulate the cycle of cell division in other areas of the body. These facts suggest that the circadian clock may regulate adult neurogenesis in the hippocampus as well. In the present study, neurogenesis in the hippocampal subgranular zone was examined in arrhythmic Bmal1 knockout (-KO) mice and their rhythmic heterozygous and wildtype littermates. Proliferation and survival of newly generated subgranular zone cells were examined using bromodeoxyuridine labelling, while pyknosis (a measure of cell death) and hippocampal volume were examined in cresyl violet stained sections. There was no significant difference in cellular proliferation between any of the groups, yet survival of proliferating cells, 6 weeks after the bromodeoxyuridine injection, was significantly greater in the BMAL1-KO animals. The number of pyknotic cells was significantly decreased in Bmal1-KO animals, yet hippocampal volume remained the same across genotypes. These findings suggest that while a functional circadian clock is not necessary for normal proliferation of neuronal precursor cells, the normal pruning of newly generated neurons in the hippocampus may require a functional circadian clock.

The expression and distribution patterns of transmitter receptors change dramatically during pre- and post-natal development of the visual cortex, but the factors that control these processes are largely unknown. We have tested the hypothesis that input activity from the lateral geniculate nucleus (LGN), one major input source to visual cortex, may contribute to the processes underlying transmitter receptor redistributions in the visual cortex during development. We found that a short period of tetrodotoxin (TTX) treatment in LGN retarded the developmental expression and age-dependent reorganization of neuromodulatory receptors, including muscarinic, serotonergic and adrenergic receptors, in kitten primary visual cortex. The visual cortices ipsilateral to the TTX infusion site displayed a 'younger' receptor pattern than that of their contralateral control counterparts in the same animals. The results suggest that active input from LGN regulates the expression profile of a broad range of receptors in the developing visual cortex.

Recent evidence suggests that the medial prefrontal cortex (MFC) is important for processing contextual information. Here we evaluate the performance of mice with MFC damage in a discrimination task that requires an association between an object and the context in which it was experienced (the object/context mismatch task), as well as a version of the novel object preference task that does not require knowledge of contextual information to resolve. Adult C57/BL6 mice received aspiration lesions of the MFC or control surgery. Upon recovery, mice were tested in the object/context mismatch and novel object preference tasks. The object/context mismatch task involved exposing mice to two different contexts, each of which housed a unique pair of identical objects. After a brief delay, mice were re-exposed to one of the contexts, this time with one object that was congruent with that context and one that was not. Novel object preference was performed within a single context, housing an identical pair of objects. After the initial exposure and following a brief delay, mice were re-exposed to the context, this time housing a familiar and a novel object. Control mice were able to successfully resolve the object/context mismatch and novel object preference discriminations, investigating the incongruent/novel object within each task significantly greater than chance. Mice with MFC damage experienced deficits in the object/context mismatch task but not the novel object preference task. These findings add to a growing body of evidence that demonstrate a critical role for the MFC in contextual information processing.

The divalent cation zinc is an essential element, having both universal and specified functions throughout the body. In the mammalian telencephalon, zinc has extensive effects on neurotransmission, affecting receptor function and second messenger systems. Through these means, it is often postulated that zinc has a fundamental role in regulating cortical synaptic function. Given that plasticity, that is, the morphological and physiological alterations that occur within neurons, is a defining characteristic of the brain, it is of particular interest to examine the mechanisms by which zinc might be involved in this process. In this review, the neurobiological characteristics of zinc will be discussed, including its distribution and the processes by which its homeostasis is regulated. As well, the substantial effects zinc may have on neuronal functioning will be examined. Finally, evidence gathered from electrophysiological, behavioural, and anatomical experiments are utilized to argue for a role of zinc in cortical plasticity.

The barrel cortex of rodents is densely innervated by a prominent subclass of glutamatergic neurons that sequester and release zinc from their synaptic boutons. These neurons may play an important role in barrel cortex function and plasticity, as zinc has been shown to modulate synaptic function by regulating neurotransmitter release, excitatory and inhibitory amino acid receptors, and second messenger signaling cascades. Here, we utilized intracortical infusions of sodium selenite to identify the source of the zincergic innervation to the mouse barrel cortex. Our results demonstrate that the majority of zincergic projections to the barrel cortex arose from ipsilateral and callosal neurons, situated in cortical layers 2/3 and 6. Regionally, these labeled neurons were most abundant within the barrel cortex itself, posterior parietal association cortex, secondary somatosensory cortex, and motor cortex. Labeled neurons were also found in other somatosensory regions corresponding to the trunk, fore- and hindlimb, as well as more distant regions such as the visual, rhinal, dorsal peduncular and insular cortices, the claustrum, and lateral and basolateral amygdaloid nuclei. Further, some mice were injected with the retrograde tracer cholera toxin subunit B to compare retrograde labeling of zincergic neurons with that of the general population of neurons innervating the barrel cortex. Our data indicate that all cortical regions providing inputs to the barrel cortex possess a zincergic component, whereas those from thalamic or brainstem structures do not. These findings demonstrate that zincergic pathways comprise a chemospecific associational network that reciprocally interconnects the barrel cortex with other cortical and limbic structures. J. Comp. Neurol. 486:48–60, 2005. © 2005 Wiley-Liss, Inc.

Synaptically released zinc is thought to play an important role in neuronal signaling by modulating excitatory and inhibitory receptors and intracellular signaling proteins. Consequently, neurons that release zinc have been implicated in synaptic plasticity underlying learning and memory as well as neuropathological processes such as epilepsy, stroke, and Alzheimer's disease. To characterize the distribution of these neurons, investigators have relied on a technique that involves the retrograde transport of zinc-selenium crystals from axonal boutons to the cell bodies of origin. However, one major problem with this method is that labeling of cell bodies is obscured by high levels of staining in synaptic boutons, particularly within forebrain structures where this staining is most intense. Here, we used a modification of the retrograde labeling method that eliminates terminal staining for zinc, thereby enabling a clear and comprehensive description of these neurons. Zincergic neurons were found in all cerebral cortical regions and were arranged in a distinct laminar pattern, restricted to layers 2/3, 5, and 6 with no labeling in layer 4. In the hippocampus, labeling was present in CA1, CA3, and the dentate gyrus but not in CA2. Labeled cell bodies were also observed in most amygdaloid nuclei, anterior olfactory nuclei, claustrum, tenia tecta, endopiriform region, lateral ventricle, lateral septum, zona incerta, superior colliculus, and periaqueductal gray. Moreover, retrograde labeling was also noted in the dorsomedial and lateral hypothalamus, regions that previously were thought to be devoid of neurons with a zincergic phenotype. Collectively these data show that zincergic neurons comprise a large population of neurons in the murine forebrain and will provide an anatomical framework for understanding the functional importance of these neurons in the mammalian brain.

Zinc is packaged in, and released from, a subset of glutamatergic synapses in the mammalian telencephalon where it has been shown to act as a potent neuromodulator. In order to establish the functional role for zincergic neurons in visual cortical function and plasticity we have compared the topographic distribution of zincergic terminals in the primary visual cortex (V1) of normal adult vervet monkeys (Cercopithicus aethiops) to that in monkeys monocularly deprived of visual input for short (24 h) or long (3 months) survival times. In normal animals, staining levels for zinc were highest in layers 1-3, 4b, 5 and 6 and lowest in layers 4a and 4c. The laminar and tangential patterns of zinc staining were complementary to staining patterns demonstrated using cytochrome oxidase (CO) histochemistry. Following 3 months of monocular deprivation by enucleation, levels of zinc staining in layers 3, 4calpha and 6a were heterogeneously reduced, clearly revealing the ocular dominance pattern in V1. When compared with the pattern of CO staining, levels of both CO and zinc were reduced in cortical territory innervated by the enucleated eye. Zinc histochemistry also revealed the ocular dominance pattern after only 24 h of monocular impulse blockade induced by enucleation or intravitreal tetrodotoxin infusion. However, by either means of deprivation for 24 h, levels of zinc were increased in deprived-eye stripes relative to nondeprived-eye stripes. These results indicate that zincergic terminals demarcate distinct compartments in the primate visual cortex. Furthermore, levels of synaptic zinc are rapidly and dynamically regulated, suggesting that zinc and/or zincergic neurons participate in mediating activity-dependent changes in the organization of the adult neocortex.

Physiological studies have shown that synaptically released zinc plays an important role in neural signaling by modulating a number of excitatory and inhibitory neurotransmitter receptors and intracellular signaling proteins. In order to localize neurons having a zincergic phenotype, Slomianka et al. [Neuroscience 38 (1990) 843] developed a labeling technique, based on the systemic administration of sodium selenite, that results in the retrograde transport of zinc-selenide crystals from axonal boutons to the cell bodies of origin. A major problem associated with this method is that the zincergic neurons are obscured by high levels of staining within synaptic boutons. In the present study, we describe a modification of the procedure for retrograde labeling of zincergic neurons, that uses a preincubation step with H2O2, which eliminates labeling of axon terminals while leaving the staining of cell bodies intact. Using this method we reveal that zincergic neurons comprise a large proportion of neurons in the murine forebrain, underscoring their contribution to network properties therein.

A goal of this study was to use recombinant adenovirus (AdV) to ectopically express Emx2 in the embryonic neocortex as a gain-of-function approach to study its role in the area-specific targeting of thalamocortical axons (TCAs), using the rat as a model. First, we cloned the cDNA for the full-length coding region of rat Emx2, a homologue of Drosophila empty spiracles. We also used this sequence to define the full-length coding region of mouse Emx2 from genomic DNA. Our analysis of Emx2 expression shows that in rat, as reported in mouse, Emx2 is expressed in high caudal to low rostral, and high medial to low lateral, gradients across the cortex throughout cortical neurogenesis, and expression is primarily restricted to progenitors in the neuroepithelium. We also carried out an analysis of the distribution of cells infected with a replication defective recombinant type 5 adenovirus (AdV) containing a CAG/LacZ expression construct, following an injection into the lateral ventricle of the cerebral hemisphere at different stages of embryonic cortical neurogenesis. AdV-infected cells are broadly distributed tangentially, but their laminar distribution is differentially restricted and reflects the temporal sequence of generation of cortical neurons. This finding indicates that the AdV predominantly infects progenitors in the ventricular zone, which leads to a preferential labeling of their immediate progeny, and infects cells that have recently become postmitotic and have yet to move far from the ventricular surface. We then show that AdV-mediated ectopic Emx2 expression results in aberrant intracortical pathfinding and areal targeting of TCAs from the dorsal lateral geniculate nucleus. These findings indicate that EMX2 imparts positional information normally associated with caudal cortical areas, such as the primary visual area, that influences the area-specific targeting of TCAs. These results are consistent with a role for EMX2 in areal specification of the neocortex as suggested by recent analyses of Emx2 null mutants.

Recent studies have identified the potential for an important role for serotonin (5-HT) receptors in the developmental plasticity of the kitten visual cortex. 5-HT 2C receptors are transiently expressed in a patchy fashion in the visual cortex of kittens between 30–80 days of age complementary to patches demarcated by cytochrome oxidase staining. 5-HT, operating via 5-HT 2C receptors, increases cortical synaptic plasticity as assessed both in brain slices and in vivo . Herein, we report that bath application of 5-HT substantially increases the probability of long-term potentiation within 5-HT 2C receptor-rich zones of cortex, but this effect is not observed in the 5-HT 2C receptor-poor zones. Instead, in these zones, 5-HT application increases the probability of long-term depression. These location-specific effects of 5-HT may promote the formation of compartment-specific cortical responses.

The electroencephalographic (EEC) activity of the hippocampus was examined in adult freely moving rats that had had all of the neocortex, including the cingulate cortex and cingulum, removed at birth. Both the cholinergic and serotonergic forms of rhythmical slow activity (RSA or theta) were present in the dorsal hippocampus and retained their normal relation to behavior. The results show that following decortication, inputs to and connections within the hippocampus apparently retain the ability to produce normal EEG. Since adult decortications abolish serotonergic RSA, the results also suggest that following neonatal injury there is reorganization within remaining serotonergic projections to the hippocampus that spare this form of RSA.

Early life events can modulate brain development to produce persistent physiological and behavioural phenotypes that are transmissible across generations. However, whether neural precursor cells are altered by early life events, to produce persistent and transmissible behavioural changes, is unknown. Here, we show that bi-parental care, in early life, increases neural cell genesis in the adult rodent brain in a sexually dimorphic manner. Bi-parentally raised male mice display enhanced adult dentate gyrus neurogenesis, which improves hippocampal neurogenesis-dependent learning and memory. Female mice display enhanced adult white matter oligodendrocyte production, which increases proficiency in bilateral motor coordination and preference for social investigation. Surprisingly, single parent-raised male and female offspring, whose fathers and mothers received bi-parental care, respectively, display a similar enhancement in adult neural cell genesis and phenotypic behaviour. Therefore, neural plasticity and behavioural effects due to bi-parental care persist throughout life and are transmitted to the next generation.

This review covers recent progress in three major areas of investigation in somatosensory systems: development, developmental plasticity and functional reorganization. Important findings relate to the development of periphery-related patterning in thalamic afferents to somatosensory cortex, the controversial role of neural activity in the development and plasticity of periphery-related afferent patterning in the brainstem and cortex, experience-dependent reorganizations in adult somatosensory cortex, and the locus of these changes.

There is considerable physiological evidence for the compartmentalization of mammalian visual cortex into functional columnar modules, representing features of visual information processing such as eye and orientation specificity. However, anatomical markers of visual cortical compartmentalization have been described only for primate visual cortex. In this report, we describe an interdigitated mosaic of four neuroactive molecules which demarcate two distinct columnar systems in the kitten visual cortex. Serotonin 1C receptors and synaptic zinc were found to demarcate columns within layer IV of kitten visual cortex, which were interdigitated with a second, patchy system characterized by increased levels of cytochrome oxidase and acetylcholinesterase. In primate visual cortex, as well as in the kitten, synaptic zinc was periodically distributed in a manner precisely complementary to cytochrome oxidase. These findings provide an anatomical framework on which unifying hypotheses of the functional organization of columnar systems in mammalian visual cortex can be built.

The ontogenic expression of the glial-specific protein S100 beta was examined in postnatal cat visual cortex using immunocytochemical methods. Astrocytes in visual cortex and oligodendrocytes in the subcortical white matter exhibited distinct spatio-temporal gradients in their expression of the S100 beta protein. In the visual cortex, S100 beta-immunoreactivity was detected in astroglial cytoplasm, as well as in the extracellular interstitium, in a lamina-specific manner throughout postnatal development. Using double labeling procedures, the S100 beta protein was found to be strictly colocalized with GFAP-immunoreactive astrocytes when GFAP was present. The glial fibrillary acidic protein (GFAP), a marker of mature astrocytes, was not present at high levels until the 4th postnatal week. From the 2nd through 5th postnatal weeks, the expression of S100 beta was highest in the thalamocortical recipient, layer IV, of visual cortical areas 17 and 18. At ages beyond 6 postnatal weeks, S100 beta-immunoreactivity increased disproportionately in supra- and infragranular layers such that areas 17 and 18 were demarcated from adjacent cortices by lower levels in layer IV. The S100 beta protein was also highly expressed in oligodendroglial somata and processes in the subcortical white matter between the 2nd and 6th postnatal weeks. The levels of S100 beta in the subcortical white matter progressively diminished to adult levels, where it was localized only to a few remaining oligodendroglial somata. The differential laminar expression of the S100 beta protein in astrocytes during the period within which the visual cortex exhibits input- and experience-dependent synaptic modifications suggests that astrocytes, possibly via their release of S100 beta, may play a special role in mediating plasticity in visual cortical development. A consistent feature of the appearance of the S100 beta protein was its expression in immature astroglia and oligodendroglia, well before they are considered morphologically mature. This characteristic underscores the potential of S100 beta as a marker of distinct populations of glial cells and of their role in normal and abnormal development.

Subcutaneous administration of the neurotoxin methylmercuric chloride to developing rats produced movement and postural disorders during the 4th postnatal week. Cytochrome oxidase histochemistry revealed an increase in the oxidative metabolic activity of small neurons within the magnocellular red nucleus (RMC) and the interrubral mesencephalon. A concurrent suppression of cytochrome oxidase activity in the large neurons and neuropil of RMC was apparent relative to controls. Decortication on postnatal day 3 did not alter the course of motor impairment or the cytochrome oxidase histopathology, suggesting that the role of neocortex in the pathogenesis of methylmercury-induced movement and postural disorders is minimal.