Induction of long-term depression and rebound potentiation by inositol trisphosphate in cerebellar Purkinje neurons

Cerebellar Purkinje neurons receive two major excitatory inputs, the climbing fibers (CFs) and parallel fibers (PFs). Simultaneous, repeated activation of CFs and PFs results in the long-term depression (LTD) of the amplitude of PF-evoked synaptic currents. To induce LTD, activation of CFs may be substituted with depolarization of the Purkinje neuron to turn on voltage-activated calcium channels and increase the intracellular calcium concentration. The role of PFs in the induction of LTD, however, is less clear. PFs activate glutamate metabotropic receptors that increase phosphoinositide turnover and elevate cytosolic inositol 1,4,5-trisphosphate (InsP3). It has been proposed that calcium release from intracellular stores via InsP3 receptors may be important in the induction of LTD. We studied the role of InsP3 in the induction of LTD by photolytic release of InsP3 from its biologically inactive “caged” precursor in voltage-clamped Purkinje neurons in acutely prepared cerebellar slices. We find that InsP3-evoked calcium release is as effective in LTD induction as activation of PFs. InsP3-induced LTD was prevented by calcium chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N′,N′-tetraacetic acid. LTD produced either by repeated activation of PFs combined with depolarization (PF+ΔV), or by InsP3 combined with depolarization (InsP3+ΔV) saturated at ≈50%. Maximal LTD induced by PF+ΔV could not be further increased by InsP3+ΔV and vice versa, which suggests that both protocols for induction of LTD share a common path. In addition to inducing LTD, photo-release of InsP3+ΔV resulted in the rebound potentiation of inhibitory synaptic currents. In the presence of heparin, an InsP3 receptor antagonist, repeated activation of PF+ΔV failed to induce LTD, suggesting that InsP3 receptors play an important role in LTD induction under physiological conditions.

Syntax processing by auditory cortical neurons in the FM–FM area of the mustached bat Pteronotus parnellii

Syntax denotes a rule system that allows one to predict the sequencing of communication signals. Despite its significance for both human speech processing and animal acoustic communication, the representation of syntactic structure in the mammalian brain has not been studied electrophysiologically at the single-unit level. In the search for a neuronal correlate for syntax, we used playback of natural and temporally destructured complex species-specific communication calls—so-called composites—while recording extracellularly from neurons in a physiologically well defined area (the FM–FM area) of the mustached bat’s auditory cortex. Even though this area is known to be involved in the processing of target distance information for echolocation, we found that units in the FM–FM area were highly responsive to composites. The finding that neuronal responses were strongly affected by manipulation in the time domain of the natural composite structure lends support to the hypothesis that syntax processing in mammals occurs at least at the level of the nonprimary auditory cortex.

Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse

Although it has been known for some time that estrogen exerts a profound influence on brain development a definitive demonstration of the role of the classical estrogen receptor (ERα) in sexual differentiation has remained elusive. In the present study we used a sexually dimorphic population of dopaminergic neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) to test the dependence of sexual differentiation on a functional ERα by comparing the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the AVPV of wild-type (WT) mice with that of mice in which the ERα had been disrupted by homologous recombination (ERKOα). Only a few ERα-immunoreactive neurons were detected in the AVPV of ERKOα mice, and the number of TH-immunoreactive neurons was three times that of WT mice, suggesting that disruption of the ERα gene feminized the number of TH-immunoreactive neurons. In contrast, the AVPV contains the same number of TH-immunoreactive neurons in testicular feminized male mice as in WT males, indicating that sexual differentiation of this population of neurons is not dependent on an intact androgen receptor. The number of TH-immunoreactive neurons in the AVPV of female ERKOα mice remained higher than that of WT males, but TH staining appeared to be lower than that of WT females. Thus, the sexual differentiation of dopamine neurons in the AVPV appears to be receptor specific and dependent on the perinatal steroid environment.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35−/− mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35−/−Cdk5 +/− mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35−/− mice compared with Cdk5−/− mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35−/− causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

β-Neuregulin-1 is required for the in vivo development of functional Ca2+-activated K+ channels in parasympathetic neurons

The development of functional Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of β-neuregulin-1. β-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger–Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic KCa in CG neurons. Injection of β-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional KCa channels at stages before the normal appearance of these channels in CG neurons developing in vivo. Conversely, injection of a neutralizing antiserum specific for β-neuregulin-1 inhibited the development of KCa channels in CG neurons. Low concentrations of β-neuregulin-1 evoked a robust increase in whole-cell KCa in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of β-neuregulin-1 by themselves was insufficient to stimulate KCa. These data suggest that the preganglionic factor required for the development of KCa in ciliary ganglion neurons is an isoform of β-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.

Differences in quantal amplitude reflect GluR4- subunit number at corticothalamic synapses on two populations of thalamic neurons

Low-frequency thalamocortical oscillations that underlie drowsiness and slow-wave sleep depend on rhythmic inhibition of relay cells by neurons in the reticular nucleus (RTN) under the influence of corticothalamic fibers that branch to innervate RTN neurons and relay neurons. To generate oscillations, input to RTN predictably should be stronger so disynaptic inhibition of relay cells overcomes direct corticothalamic excitation. Amplitudes of excitatory postsynaptic conductances (EPSCs) evoked in RTN neurons by minimal stimulation of corticothalamic fibers were 2.4 times larger than in relay neurons, and quantal size of RTN EPSCs was 2.6 times greater. GluR4-receptor subunits labeled at corticothalamic synapses on RTN neurons outnumbered those on relay cells by 3.7 times, providing a basis for differences in synaptic strength.

F-spondin is a contact-repellent molecule for embryonic motor neurons

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural–functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.

The generation, migration, and differentiation of olfactory neurons in the adult primate brain

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results—obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers—indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale.

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP+ cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP+ cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.