Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1A-subunit

The Ca2+ channel α1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. α1A-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. We generated α1A-deficient mice (α1A−/−) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying ≈3–4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in α1A−/− hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca2+ entry. The α1A−/− mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in α1A.

Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of ataxia–telangiectasia

Neural degeneration is one of the clinical manifestations of ataxia–telangiectasia, a disorder caused by mutations in the Atm protein kinase gene. However, neural degeneration was not detected with general purpose light microscopic methods in previous studies using several different lines of mice with disrupted Atm genes. Here, we show electron microscopic evidence of degeneration of several different types of neurons in the cerebellar cortex of 2-month-old Atm knockout mice, which is accompanied by glial activation, deterioration of neuropil structure, and both pre- and postsynaptic degeneration. These findings are similar to those in patients with ataxia–telangiectasia, indicating that Atm knockout mice are a useful model to elucidate the mechanisms underlying neurodegeneration in this condition and to develop and test strategies to palliate and prevent the disease.

Glutamate transporter currents in Bergmann glial cells follow the time course of extrasynaptic glutamate

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate flux. Although a complete cycle of transport may require 50–100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of l-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5′ untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

Sodium channel Nav1.6 is localized at nodes of Ranvier, dendrites, and synapses

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Nav1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Nav1.6-deficient mouse mutant strain med, whose nodes were negative for Nav1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Nav1.6 is the predominant channel type in this structure. In the central nervous system, Nav1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Nav1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Postmortem prefrontal cortices (PFC) (Brodmann’s areas 10 and 46), temporal cortices (Brodmann’s area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (≈50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from γ-aminobutyric acid (GABA)A receptors α1 and α5 and nicotinic acetylcholine receptor α7 subunits. Whereas the expression of the α7 nicotinic acetylcholine receptor subunit was normal, that of the α1 and α5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of ≈50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability “two-hit” model for the etiology of schizophrenia.

Widespread expression and estrogen regulation of PPEIA-3′ nuclear RNA in the rat brain

We previously identified a novel nuclear RNA species derived from the preproenkephalin (PPE) gene. This transcript, which we have named PPEIA-3′ RNA, hybridizes with probes directed at a region of PPE intron A downstream of an alternative germ-cell transcription start site, but does not contain PPE protein coding sequences. We now report that estrogen treatment of ovariectomized rats increases the expression of conventional PPE heteronuclear RNA, and also induces the expression of PPEIA-3′ RNA, apparently in separate cell populations within the ventromedial nucleus of the hypothalamus. Further, we show that cells expressing PPEIA-3′ are found in several neuronal groups in the rat forebrain and brainstem, with a distinct topographical distribution. High densities of PPEIA-3′ containing cells are found in the reticular thalamic nucleus, the basal forebrain, the vestibular complex, the deep cerebellar nuclei, and the trapezoid body, a pattern that parallels the distribution of atypical nuclear RNAs described by other groups. These results suggest that this diverse neuronal population shares a common set of nuclear factors responsible for the expression and retention of this atypical RNA transcript. The implication of these results for cell-specific gene transcription and regulation in the brain and the possible relationship of PPEIA-3′ RNA and other atypical nuclear RNAs is discussed.

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.

Neuroimaging evidence implicating cerebellum in the experience of hypercapnia and hunger for air

Recent neuroimaging and neurological data implicate cerebellum in nonmotor sensory, cognitive, vegetative, and affective functions. The present study assessed cerebellar responses when the urge to breathe is stimulated by inhaled CO2. Ventilation changes follow arterial blood partial pressure CO2 changes sensed by the medullary ventral respiratory group (VRG) and hypothalamus, entraining changes in midbrain, pons, thalamus, limbic, paralimbic, and insular regions. Nearly all these areas are known to connect anatomically with the cerebellum. Using positron emission tomography, we measured regional brain blood flow during acute CO2-induced breathlessness in humans. Separable physiological and subjective effects (air hunger) were assessed by comparisons with various respiratory control conditions. The conjoint physiological effects of hypercapnia and the consequent air hunger produced strong bilateral, near-midline activations of the cerebellum in anterior quadrangular, central, and lingula lobules, and in many areas of posterior quadrangular, tonsil, biventer, declive, and inferior semilunar lobules. The primal emotion of air hunger, dissociated from hypercapnia, activated midline regions of the central lobule. The distributed activity across the cerebellum is similar to that for thirst, hunger, and their satiation. Four possible interpretations of cerebellar function(s) here are that: it subserves implicit intentions to access air; it provides predictive internal models about the consequences of CO2 inhalation; it modulates emotional responses; and that while some cerebellar regions monitor sensory acquisition in the VRG (CO2 concentration), others influence VRG to adjust respiratory rate to optimize partial pressure CO2, and others still monitor and optimize the acquisition of other sensory data in service of air hunger aroused vigilance.

An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis

Programmed cell death (PCD) during neuronal development and disease has been shown to require de novo RNA synthesis. However, the time course and regulation of target genes is poorly understood. By using a brain-biased array of over 7,500 cDNAs, we profiled this gene expression component of PCD in cerebellar granule neurons challenged separately by potassium withdrawal, combined potassium and serum withdrawal, and kainic acid administration. We found that hundreds of genes were significantly regulated in discreet waves including known genes whose protein products are involved in PCD. A restricted set of genes was regulated by all models, providing evidence that signals inducing PCD can regulate large assemblages of genes (of which a restricted subset may be shared in multiple pathways).

An unliganded thyroid hormone receptor causes severe neurological dysfunction

Congenital hypothyroidism and the thyroid hormone (T3) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T3 binding mutation was introduced into the mouse TR-β locus by homologous recombination. Because of this T3 binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.

Can medial temporal lobe regions distinguish true from false? An event-related functional MRI study of veridical and illusory recognition memory

To investigate the types of memory traces recovered by the medial temporal lobe (MTL), neural activity during veridical and illusory recognition was measured with the use of functional MRI (fMRI). Twelve healthy young adults watched a videotape segment in which two speakers alternatively presented lists of associated words, and then the subjects performed a recognition test including words presented in the study lists (True items), new words closely related to studied words (False items), and new unrelated words (New items). The main finding was a dissociation between two MTL regions: whereas the hippocampus was similarly activated for True and False items, suggesting the recovery of semantic information, the parahippocampal gyrus was more activated for True than for False items, suggesting the recovery of perceptual information. The study also yielded a dissociation between two prefrontal cortex (PFC) regions: whereas bilateral dorsolateral PFC was more activated for True and False items than for New items, possibly reflecting monitoring of retrieved information, left ventrolateral PFC was more activated for New than for True and False items, possibly reflecting semantic processing. Precuneus and lateral parietal regions were more activated for True and False than for New items. Orbitofrontal cortex and cerebellar regions were more activated for False than for True items. In conclusion, the results suggest that activity in anterior MTL regions does not distinguish True from False, whereas activity in posterior MTL regions does.

Memory systems in the brain and localization of a memory

It is now clear that there are a number of different forms or aspects of learning and memory that involve different brain systems. Broadly, memory phenomena have been categorized as explicit or implicit. Thus, explicit memories for experience involve the hippocampus–medial temporal lobe system and implicit basic associative learning and memory involves the cerebellum, amygdala, and other systems. Under normal conditions, however, many of these brain–memory systems are engaged to some degree in learning situations. But each of these brain systems is learning something different about the situation. The cerebellum is necessary for classical conditioning of discrete behavioral responses (eyeblink, limb flexion) under all conditions; however, in the “trace” procedure where a period of no stimuli intervenes between the conditioned stimulus and the unconditioned stimulus the hippocampus plays a critical role. Trace conditioning appears to provide a simple model of explicit memory where analysis of brain substrates is feasible. Analysis of the role of the cerebellum in basic delay conditioning (stimuli overlap) indicates that the memories are formed and stored in the cerebellum. The phenomenon of cerebellar long-term depression is considered as a putative mechanism of memory storage.

The effects of practice on the functional anatomy of task performance

The effects of practice on the functional anatomy observed in two different tasks, a verbal and a motor task, are reviewed in this paper. In the first, people practiced a verbal production task, generating an appropriate verb in response to a visually presented noun. Both practiced and unpracticed conditions utilized common regions such as visual and motor cortex. However, there was a set of regions that was affected by practice. Practice produced a shift in activity from left frontal, anterior cingulate, and right cerebellar hemisphere to activity in Sylvian-insular cortex. Similar changes were also observed in the second task, a task in a very different domain, namely the tracing of a maze. Some areas were significantly more activated during initial unskilled performance (right premotor and parietal cortex and left cerebellar hemisphere); a different region (medial frontal cortex, “supplementary motor area”) showed greater activity during skilled performance conditions. Activations were also found in regions that most likely control movement execution irrespective of skill level (e.g., primary motor cortex was related to velocity of movement). One way of interpreting these results is in a “scaffolding-storage” framework. For unskilled, effortful performance, a scaffolding set of regions is used to cope with novel task demands. Following practice, a different set of regions is used, possibly representing storage of particular associations or capabilities that allow for skilled performance. The specific regions used for scaffolding and storage appear to be task dependent.