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Human cortical excitability can be modified by repetitive transcranial magnetic stimulation (rTMS), but the cellular mechanisms are largely unknown. Here, we show that the pattern of delivery of theta-burst stimulation (TBS) (continuous versus intermittent) differently modifies electric activity and protein expression in the rat neocortex. Intermittent TBS (iTBS), but not continuous TBS (cTBS), enhanced spontaneous neuronal firing and EEG gamma band power. Sensory evoked cortical inhibition increased only after iTBS, although both TBS protocols increased the first sensory response arising from the resting cortical state. Changes in the cortical expression of the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) indicate that changes in spontaneous and evoked cortical activity following rTMS are in part related to altered activity of inhibitory systems. By reducing PV expression in the fast-spiking interneurons, iTBS primarily affected the inhibitory control of pyramidal cell output activity, while cTBS, by reducing CB expression, more likely affected the dendritic integration of synaptic inputs controlled by other classes of inhibitory interneurons. Calretinin, the third major calcium-binding protein expressed by another class of interneurons was not affected at all. We conclude that different patterns of TBS modulate the activity of inhibitory cell classes differently, probably depending on the synaptic connectivity and the preferred discharge pattern of these inhibitory neurons.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.

Cerebral cortical functions are conducted by two general classes of neurons: glutamatergic projection neurons and GABAergic interneurons. Distinct interneuron subtypes serve distinct roles in modulating cortical activity and can be differentially affected in cortical diseases, but little is known about the mechanisms for generating their diversity. Recent evidence suggests that many cortical interneurons originate within the subcortical telencephalon and then migrate tangentially into the overlying cortex. To test the hypothesis that distinct interneuron subtypes are derived from distinct telencephalic subdivisions, we have used an in vitro assay to assess the developmental potential of subregions of the telencephalic proliferative zone (PZ) to give rise to neurochemically defined interneuron subgroups. PZ cells from GFP+ donor mouse embryos were transplanted onto neonatal cortical feeder cells and assessed for their ability to generate specific interneuron subtypes. Our results suggest that the parvalbumin- and the somatostatin-expressing interneuron subgroups originate primarily within the medial ganglionic eminence (MGE) of the subcortical telencephalon, whereas the calretinin-expressing interneurons appear to derive mainly from the caudal ganglionic eminence (CGE). These results are supported by findings from primary cultures of cortex from Nkx2.1 mutants, in which normal MGE fails to form but in which the CGE is less affected. In these cultures, parvalbumin- and somatostatin-expressing cells are absent, although calretinin-expressing interneurons are present. Interestingly, calretinin-expressing bipolar interneurons were nearly absent from cortical cultures of Dlx1/2 mutants. By establishing spatial differences in the origins of interneuron subtypes, these studies lay the groundwork for elucidating the molecular bases for their distinct differentiation pathways.

The role of the suprachiasmatic nuclei (SCN) in generating circadian rhythms in physiology and behavior is well established. Recent evidence based on clock gene expression indicates that the rodent SCN are composed of at least two functional subdivisions. In Syrian hamsters (Mesocricetus auratus), cells in a subregion of the caudal SCN marked by calbindin-D(28K) (CalB) express light-induced, but not rhythmic, clock genes (Per1, Per2, and Per3). In the SCN region marked by vasopressinergic cells and fibers, clock gene expression is rhythmic. Importantly, lesions of the CalB subregion that spare a significant portion of the SCN abolish rhythms in locomotor behavior. One possibility is that the CalB subregion is required to maintain SCN function necessary to support all behavioral and physiological rhythms. Alternatively, this subregion may control circadian rhythms in locomotor behavior, whereas other circadian responses in physiology and behavior are sustained by different SCN compartments. The present study sought to distinguish between these possibilities by examining the role of the CalB subregion in a battery of rhythms within an individual animal. The results indicate that lesions of the CalB subregion of the SCN abolish circadian rhythms in behavior (locomotion, drinking, gnawing), physiology (body temperature, heart rate), and hormone secretion (melatonin, cortisol), even when other SCN compartments are spared. Together, these findings suggest a novel fundamental property of SCN organization, with a subset of cells being critical for the maintenance of SCN function manifest in circadian rhythms in physiology and behavior.

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Na(v)1.3 (SCN3) Na channel isoform.

Many of the interneuron cell types present in the adult spinal cord contribute to the circuits that control locomotion and posture. Little is known, however, about the embryonic origin of these cell types or the molecular mechanisms that control their differentiation. Here we provide evidence that V1 interneurons (INs), an embryonic class of interneurons that transiently express the En1 transcription factor, differentiate as local circuit inhibitory interneurons and form synapses with motor neurons. Furthermore, we show that a subset of V1 INs differentiates as Renshaw cells, the interneuronal cell type that mediates recurrent inhibition of motor neurons. We analyze the role that two V1 IN-related transcription factor genes play in Renshaw cell development. Pax6 (paired box gene 6) is necessary for an early step in Renshaw cell development, whereas Engrailed 1 (En1), which is genetically downstream of Pax6, regulates the formation of inhibitory synapses between Renshaw cells and motor neurons. Together, these results show that Pax6 and En1 have essential roles in establishing the recurrent inhibitory circuit between motor neurons and Renshaw cells.

Processing of visual information is performed in different cortical areas that are interconnected by feedforward (FF) and feedback (FB) pathways. Although FF and FB inputs are excitatory, their influences on pyramidal neurons also depend on the outputs of GABAergic neurons, which receive FF and FB inputs. Rat visual cortex contains at least three different families of GABAergic neurons that express parvalbumin (PV), calretinin (CR), and somatostatin (SOM) (Gonchar and Burkhalter, 1997). To examine whether pathway-specific inhibition (Shao and Burkhalter, 1996) is attributable to distinct connections with GABAergic neurons, we traced FF and FB inputs to PV, CR, and SOM neurons in layers 1-2/3 of area 17 and the secondary lateromedial area in rat visual cortex. We found that in layer 2/3 maximally 2% of FF and FB inputs go to CR and SOM neurons. This contrasts with 12-13% of FF and FB inputs onto layer 2/3 PV neurons. Unlike inputs to layer 2/3, connections to layer 1, which contains CR but lacks SOM and PV somata, are pathway-specific: 21% of FB inputs go to CR neurons, whereas FF inputs to layer 1 and its CR neurons are absent. These findings suggest that FF and FB influences on layer 2/3 pyramidal neurons mainly involve disynaptic connections via PV neurons that control the spike outputs to axons and proximal dendrites. Unlike FF input, FB input in addition makes a disynaptic link via CR neurons, which may influence the excitability of distal pyramidal cell dendrites in layer 1.

The dystrophin-associated protein complex (DPC), comprising sarcoglycans, dystroglycans, dystrobrevins, and syntrophins, is a component of synapses both in muscle and brain. Dysbindin is a novel component of the DPC, which binds to beta-dystrobrevin and may serve as an adaptor protein that links the DPC to an intracellular signaling cascade. Disruption of the DPC results in muscular dystrophy, and mutations in the human ortholog of dysbindin have been implicated in the pathogenesis of schizophrenia. In both cases, patients also present with neurological symptoms reminiscent of cerebellar problems. In the mouse cerebellum, dysbindin immunoreactivity is expressed at high levels in a subset of mossy fiber synaptic glomeruli in the granular layer. Lower levels of dysbindin immunoreactivity are also detected in Purkinje cell dendrites. In the cerebellar vermis, dysbindin-immunoreactive glomeruli are restricted to an array of parasagittal stripes that bears a consistent relationship to Purkinje cell parasagittal band boundaries as defined by the expression of the respiratory isoenzyme zebrin II/aldolase c. In a mouse model of Duchenne muscular dystrophy, the mdx mutant, in which dystrophin is not expressed, there is a dramatic increase in the number of dysbindin-immunoreactive glomeruli in the posterior cerebellar vermis. Moreover, the topography of the terminal fields is disrupted, replacing the stripes by a homogeneous distribution. Abnormal synaptic organization in the cerebellum may contribute to the neurological problems associated with muscular dystrophy and schizophrenia.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

We have used time-lapse multiphoton microscopy to map the migration and settling pattern of GABAergic interneurons that originate in the ganglionic eminence of the ventral forebrain and incorporate into the neocortex of the cerebral hemispheres. Imaging of the surface of the cerebral hemispheres in both explant cultures and brains of living mouse embryos revealed that GABAergic interneurons migrating within the marginal zone originate from three different sources and migrate via distinct and independent streams. After reaching their areal destination, interneurons descend into the underlying cortex to assume positions with isochronically generated, radially derived neurons. The dynamics and pattern of cell migration in the marginal zone (see movies, available at www.jneurosci.org) suggest that the three populations of interneurons respond selectively to distinct local cues for directing their migration to the appropriate areas and layers of the neocortex. This approach opens a new avenue for study of normal and abnormal neuronal migration in their native environment and indicate that interneurons have specific programs for their areal and laminar deployment.

Afferents from the amygdala help to define the ventral striatum and mediate goal-directed behaviors. In addition to well known inputs to the classic ventral striatum, the amygdala also projects to the caudoventral striatum and amygdalostriatal area. We examined whether the primate caudoventral striatum and amygdalostriatal area can be considered part of the "ventral" striatum based on cellular and histochemical features found in the classic rostral ventral striatum. We used several histochemical stains, including calbindin-D28k, a marker of the shell compartment, acetylcholinesterase, substance P, tyrosine hydroxylase, and Bcl-2, a marker of immature neurons, to examine this question. Our results indicate that the lateral amygdalostriatal area and caudoventral striatum are "striatal like" based on intermediate to high acetylcholinesterase and tyrosine hydroxylase levels. The lateral amygdalostriatal area is chemically similar to the shell, whereas the caudoventral striatum more closely resembles the striatum outside the shell. In contrast, the medial amygdalostriatal area is more related to the central amygdaloid nucleus than to the striatum. Bcl-2 immunoreactivity is associated with granular islands and medium-sized cells in the vicinity of the ventral striatum both rostrally and caudally. Together, the caudal ventral striatum has a histochemical and cellular organization similar to that of the rostral ventral striatum, consistent with their common innervation by the amygdala and other ventral structures. In addition, Bcl-2 is expressed in and near both poles of the ventral striatum, suggesting that these areas maintain a heightened capacity for growth and plasticity compared with other striatal sectors.

Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

Granule cell precursors in the external germinal layer (EGL) of the cerebellum have been proposed to be a major player in the migration and positioning of Purkinje cells through the expression of the Netrin-like receptor Unc5h3 and the extracellular matrix molecule Reelin. To explore the role of the EGL on these processes, we made use of the math1 null-mutant mouse in which the EGL does not form. In the absence of the EGL, we find three populations of ectopic Purkinje cells. First, we find 1% of all Purkinje cells in a supracerebellar position at the dorsal midline. Second, we find 7% of all Purkinje cells in the inferior colliculus, similar to what is seen in the Unc5h3 mutant. Our finding that Unc5h3 expression is not disrupted in these cells supports the proposed role of EGL granule cell precursors in establishing the anterior cerebellar boundary through the expression of Unc5h3. Third, we find 20% of all Purkinje cells positioned deep to the cerebellar cortex as seen in the reeler mutant. However, unlike the reeler mutant, where 5% of the Purkinje cells migrate successfully, we find that in the math1 null that 72% of the Purkinje cells migrate successfully. This finding demonstrates that Purkinje cell migration is not solely dependent on Reelin signaling from the EGL and is likely caused by Reelin signals emanating from the nuclear transitory zone or the ventricular zone, or both.

Dopaminergic (DA) midbrain neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are involved in various brain functions such as voluntary movement and reward and are targets in disorders such as Parkinson's disease and schizophrenia. To study the functional properties of identified DA neurons in mouse midbrain slices, we combined patch-clamp recordings with either neurobiotin cell-filling and triple labeling confocal immunohistochemistry, or single-cell RT-PCR. We discriminated four DA subpopulations based on anatomical and neurochemical differences: two calbindin D28-k (CB)-expressing DA populations in the substantia nigra (SN/CB+) or ventral tegmental area (VTA/CB+), and respectively, two calbindin D28-k negative DA populations (SN/CB-, VTA/CB-). VTA/CB+ DA neurons displayed significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with other DA neurons. In contrast, all four DA populations possessed significant differences in I(h) channel densities and I(h) channel-mediated functional properties like sag amplitudes and rebound delays in the following order: SN/CB- --> VTA/CB- --> SN/CB+ --> VTA/CB+. Single-cell RT-multiplex PCR experiments demonstrated that differential calbindin but not calretinin expression is associated with differential I(h) channel densities. Only in SN/CB- DA neurons, however, I(h) channels were actively involved in pacemaker frequency control. In conclusion, diversity within the DA system is not restricted to distinct axonal projections and differences in synaptic connectivity, but also involves differences in postsynaptic conductances between neurochemically and topographically distinct DA neurons.

De novo steroidogenesis from cholesterol is a conserved property of vertebrate brains, and such steroids synthesized de novo in the brain are called neurosteroids. The identification of neurosteroidogenic cells is essential to the understanding of the physiological role of neurosteroids in the brain. We have demonstrated recently that neuronal neurosteroidogenesis occurs in the brain and indicated that the Purkinje cell actively synthesizes several neurosteroids de novo from cholesterol in vertebrates. Interestingly, in the rat, this neuron actively synthesizes progesterone de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs most markedly. Therefore, in this study, the possible organizing actions of progesterone during cerebellar development have been examined. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone promotes dose-dependent dendritic outgrowth of Purkinje cells but dose not affect their somata. This effect was blocked by the anti-progestin RU 486 [mifepristone; 17beta-hydroxy-11beta-(4-methylaminophenyl)-17alpha-(1-propynyl) estra-4,9-dien-3 one-6-7]. In vivo administration of progesterone to pups further revealed an increase in the density of Purkinje spine synapses electron microscopically. In contrast to progesterone, there was no significant effect of 3alpha,5alpha-tetrahydroprogesterone, a progesterone metabolite, on Purkinje cell development. Reverse transcription-PCR-Southern and immunocytochemical analyses showed that intranuclear progesterone receptors were expressed in Purkinje cells. These results suggest that progesterone promotes both dendritic outgrowth and synaptogenesis in Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development. Such organizing actions may contribute to the formation of the cerebellar neuronal circuit.

Sensory information, relayed through the thalamus, arrives in the neocortex as excitatory input, but rapidly induces strong disynaptic inhibition that constrains the cortical flow of excitation both spatially and temporally. This feedforward inhibition is generated by intracortical interneurons whose precise identity and properties were not known. To characterize interneurons generating feedforward inhibition, neurons in layers IV and V of mouse somatosensory ("barrel") cortex in vitro were tested in the cell-attached configuration for thalamocortically induced firing and in the whole-cell mode for synaptic responses. Identification as inhibitory or excitatory neurons was based on intrinsic firing patterns and on morphology revealed by intracellular staining. Thalamocortical stimulation evoked action potentials in approximately 60% of inhibitory interneurons but in <5% of excitatory neurons. The inhibitory interneurons that fired received fivefold larger thalamocortical inputs compared with nonfiring inhibitory or excitatory neurons. Thalamocortically evoked spikes in inhibitory interneurons followed at short latency the onset of excitatory monosynaptic responses in the same cells and slightly preceded the onset of inhibitory responses in nearby neurons, indicating their involvement in disynaptic inhibition. Both nonadapting (fast-spiking) and adapting (regular-spiking) inhibitory interneurons fired on thalamocortical stimulation, as did interneurons expressing parvalbumin, calbindin, or neither calcium-binding protein. Morphological analysis revealed that some interneurons might generate feedforward inhibition within their own layer IV barrel, whereas others may convey inhibition to upper layers, within their own or in adjacent columns. We conclude that feedforward inhibition is generated by diverse classes of interneurons, possibly serving different roles in the processing of incoming sensory information.

The functional properties of AMPA-type glutamate receptors are determined by their subunit composition. We detected the expression of the AMPA receptor subunits (GluR1-GluR4) in neurons in the somatosensory cortex of adult rats by combining nonradioactive in situ hybridization using digoxigenin-labeled RNA probes of GluR1 and GluR2 with immunocytochemistry using specific antibodies against GluR1, GluR2/3, and GluR4. On the basis of differential expression of the GluR1 and GluR2 subunits, we classified the cortical neurons into four categories. To correlate the differential expression of AMPA receptor subunits in each neuron with that of two calcium-binding proteins, parvalbumin and calbindin-D28k, we used a triple-labeling method. The majority of cortical neurons ( approximately 2/3) showed expression of GluR2 and undetectable expression of GluR1. GluR1-/GluR2-expressing neurons and GluR1-expressing/GluR2-undetectable neurons comprised approximately 1/10 each. Regarding the morphology, most GluR1-undetectable/GluR2-expressing neurons were pyramidal cells in layers II/III, V, and VI, whereas most GluR1-expressing/GluR2-undetectable neurons were nonpyramidal cells in layers II-VI. The GluR1-/GluR2-expressing neurons were either pyramidal or nonpyramidal. The majority of GluR1-/GluR2-expressing nonpyramidal cells was intensely stained with monoclonal antibody against calbindin-D28k, and one-half of the GluR1-undetectable/GluR2-expressing pyramidal neurons in layer II/III were lightly stained with this antibody. Most of GluR1-expressing/GluR2-undetectable neurons possessed parvalbumin immunoreactivity. These results indicate that neurons in the rat somatosensory cortex express differential combinations of GluR subunits, which correlate with the specific expression of the calcium-binding proteins.