Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin؊/؊ mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin؊/؊ mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin؊/؊ mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin؊/؊ mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and ␣-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin؊/؊ brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice. Parkinson's disease (PD)1 is an age-related movement disorder characterized by bradykinesia, rigidity, resting tremor, and postural instability. The neuropathologic hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra (SN) and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. The clinical manifestations of PD are due to progressive degeneration of dopaminergic neurons in the pars compacta of the SN that give rise to the nigrostriatal pathway, causing dopamine (DA) depletion in the striatum, where it is required for normal motor function. Little is known about the mechanisms of PD pathogenesis and nigral degeneration, although DA neurons have been shown to be susceptible to oxidative stress (1), mitochondrial defects (2), and environmental toxins (3).The recent identification of genes linked to familial forms of PD (FPD) makes it possible to investigate the pathogenic mechanism by employing genetic approaches (4 -6). Over fifty recessively inherited mutations, including deletion, frameshift, nonsense, and missense mutations, have been identified in parkin in large numbers of families, making parkin the major gene responsible for early-onset FPD (7-10). Although the first report linked parkin mutations to autosomal recessive juvenile parkinsonism (AR-JP) with atypical clinical features (5), many more cases identified subsequently were considered typical early-onset FPD with symptoms often indistinguishable from sporadic PD (9, 11). Autopsies of limited numbers of patients showed selective loss of dopaminergic neurons in the SN either in the absence (12-15) or in the ...

We examined passive and active membrane properties and synaptic responses of medium-sized spiny striatal neurons in brain slices from presymptomatic (approximately 40 days of age) and symptomatic (approximately 90 days of age) R6/2 transgenics, a mouse model of Huntington's disease (HD) and their age-matched wild-type (WT) controls. This transgenic expresses exon 1 of the human HD gene with approximately 150 CAG repeats and displays a progressive behavioral phenotype associated with numerous neuronal alterations. Intracellular recordings were obtained using standard techniques from R6/2 and age-matched WT mice. Few electrophysiological changes occurred in striatal neurons from presymptomatic R6/2 mice. The changes in this age group were increased neuronal input resistance and lower stimulus intensity to evoke action potentials (rheobase). Symptomatic R6/2 mice exhibited numerous electrophysiological alterations, including depolarized resting membrane potentials, increased input resistances, decreased membrane time constants, and alterations in action potentials. Increased stimulus intensities were required to evoke excitatory postsynaptic potentials (EPSPs) in neurons from symptomatic R6/2 transgenics. These EPSPs had slower rise times and did not decay back to baseline by 45 ms, suggesting a more prominent component mediated by activation of N-methyl-D-aspartate receptors. Neurons from both pre- and symptomatic R6/2 mice exhibited reduced paired-pulse facilitation. Data from biocytin-filled or Golgi-impregnated neurons demonstrated decreased dendritic spine densities, smaller diameters of dendritic shafts, and smaller dendritic fields in symptomatic R6/2 mice. Taken together, these findings indicate that passive and active membrane and synaptic properties of medium-sized spiny neurons are altered in the R6/2 transgenic. These physiological and morphological alterations will affect communication in the basal ganglia circuitry. Furthermore, they suggest areas to target for pharmacotherapies to alleviate and reduce the symptoms of HD.

We used two mouse models of Huntington's disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141-157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat "knockin," consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N-methyl-D-aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD.

The mechanisms responsible for seizure generation in cortical dysplasia (CD) are unknown, but morphologically abnormal cells could contribute. We examined the passive and active membrane properties of cells from pediatric CD in vitro. Normal- and abnormal-appearing cells were identified morphologically by using infrared videomicroscopy and biocytin in slices from children with mild to severe CD. Electrophysiological properties were assessed with patch clamp recordings. Four groups of abnormal-appearing cells were observed. The first consisted of large, pyramidal cells probably corresponding to cytomegalic neurons. Under conditions that reduced the contribution of K(+) conductances, these cells generated large Ca(2+) currents and influx when depolarized. When these cells were acutely dissociated, peak Ca(2+) currents and densities were greater in cytomegalic compared with normal-appearing pyramidal neurons. The second group included large, nonpyramidal cells with atypical somatodendritic morphology that could correspond to "balloon" cells. These cells did not display active voltage- or ligand-gated currents and did not appear to receive synaptic inputs. The third group included misoriented and dysmorphic pyramidal neurons, and the fourth group consisted of immature-looking pyramidal neurons. Electrophysiologically, neurons in these latter two groups did not display significant abnormalities when compared with normal-appearing pyramidal neurons. We conclude that there are cells with abnormal intrinsic membrane properties in pediatric CD. Among the four groups of cells, the most abnormal electrophysiological properties were displayed by cytomegalic neurons and large cells with atypical morphology. Cytomegalic neurons could play an important role in the generation of epileptic activity.

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.

1 The action of analogues and C-terminal fragments of neuropeptide Y (NPY) was examined on excitatory synaptic transmission in area CAl of the rat hippocampal slice in vitro, by use of intracellular and extracellular recordings, to determine by agonist profile the NPY receptor subtype mediating presynaptic inhibition. 2 Neither NPY, analogues nor fragments of NPY affected the passive or active properties of the postsynaptic CAL pyramidal neurones, indicating their action is at a presynaptic site. 3 The full-sequence analogues, peptide YY (PYY) and human NPY (hNPY), were equipotent with NPY at the presynaptic receptor, while desamido hNPY was without activity. 4 NPY2-36 was equipotent with NPY. Fragments as short as NPY13-36 were active, but gradually lost activity with decreasing length. NPY16-36 had no effect on extracellular field potentials, but still significantly inhibited excitatory postsynaptic potential amplitudes. Fragments shorter than NPY16-36 had no measurable effect on synaptic transmission. 5 The presynaptic NPY receptor in hippocampal CAL therefore shares an identical agonist profile with the presynaptic Y2 receptor at the peripheral sympathetic neuroeffector junction.

Neuropeptide Y (NPY) reduces excitatory synaptic transmission between stratum radiatum and CA1 pyramidal cells in rat hippocampal slice in vitro by a presynaptic action. To understand NPY's role in the control of excitability in hippocampus, its actions on excitatory and inhibitory synaptic transmission were examined, using intracellular, sharp microelectrode, and tight-seal, whole cell recordings from principal neurons in areas CA1, CA3, and dentate. Bath application of 1 microM NPY reversibly inhibited excitatory postsynaptic potentials (EPSPs) evoked in CA1 pyramidal cells from either stratum radiatum or stratum oriens by about 50%. Neuropeptide Y also inhibited EPSPs at mossy fiber-CA3, stratum oriens-CA3, and CA3-CA3 synapses by between 45% and 55%. As in CA1, the action of NPY was presynaptic. By contrast, NPY did not inhibit EPSPs evoked in dentate granule cells from either perforant path or commissural inputs. Neuropeptide Y did not alter postsynaptic membrane properties in any cell type. Although NPY attenuated the orthodromically evoked (stratum radiatum) inhibitory postsynaptic potentials in CA1 pyramidal cells by about the same amount as it inhibited the EPSPs, it did not affect the IPSPs evoked in the same cells by antidromic stimulation from alveus. Inhibitory postsynaptic potentials evoked in pharmacological isolation in CA1, CA3, or dentate were also not significantly affected by NPY. The evidence supports the hypothesis that NPY acts at feedforward excitatory synapses to presynaptically reduce the amplitude of excitation as it travels through hippocampal circuits. By contrast, synaptically mediated inhibition is not directly affected by NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y (NPY) potently inhibits glutamate-mediated synaptic transmission in areas CA1 and CA3 of the rat hippocampus without affecting other synaptic inputs onto principal cells of the hippocampal formation, suggesting that its biological role may include the regulation of excitability within the hippocampus. Here we examine NPY's actions in three in vitro models of epilepsy [0 Mg2+-, picrotoxin-, and stimulus-train-induced bursting (STIB)] with the use of extracellular and whole cell patch-clamp recordings from rat hippocampal-entorhinal cortex slices. Perfusion of the slice with saline that had Mg2+ omitted (0 Mg2+) or that had picrotoxin (100 microM) added resulted in brief spontaneous bursts (SBs) resembling interictal discharges. SB frequency is significantly reduced in both models by 1 microM NPY and by the Y2-preferring agonists peptide (P)YY(3-36) (1 microM) and 1-4-(6-aminohexanoic acid)-25-36 ([ahx(5-24)] NPY; 3 microM). The Y1-preferring agonist Leu31-Pro34NPY (1 microM) is considerably less potent, but also reduces burst frequency, even in the presence of the selective Y1 receptor antagonist GR231118, suggesting the involvement of a different receptor. In STIB, high-frequency stimulus trains to stratum radiatum of area CA2/CA3 result in clonic or tonic-clonic ictaform primary afterdischarges (primary ADs) as well as longer, spontaneous secondary ictaform discharges and SBs similar to those in the other models. Primary AD duration is greatly reduced or abolished by Y2- but not Y1-preferring agonists. SBs, although variable, were inhibited by both Y1 and Y2 agonists. In single and dual whole cell recordings from CA3 pyramidal cells, we frequently observed spontaneous, rhythmic synchronous events (SRSEs) arising after several STIB stimuli. Once established, SRSEs persist in the absence of further stimuli and are insensitive to the application of NPY. SRSEs in pyramidal cells typically occur at 2-4 Hz, are outward currents when cells are clamped near rest (>100 pA at a holding potential of -55 mV), reverse between -60 and -70 mV, and are inhibited by 100 microM picrotoxin, indicating involvement of gamma-aminobutyric acid-A receptors. They are inhibited by blockers of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) but not N-methyl-D-aspartate receptors. Whole cell patch-clamp recordings from interneurons in CA3 after STIB reveal NPY-insensitive, rhythmic, inward AMPA-receptor-mediated currents that are similar in frequency to SRSEs seen in pyramidal cells. We conclude that NPY, acting predominantly via Y2 receptors, can dramatically inhibit epileptiform activity in three fundamentally different in vitro models of epilepsy without affecting endogenous inhibitory activity. The results also provide support for the hypothesis that endogenous NPY may normally control excitability in the hippocampus and suggest the potential for NPY receptors as targets for anticonvulsant therapy.