Jorge Flores-Hernandez

NMDA receptor function in mouse models of Huntington disease

Huntington disease (HD) is an autosomal dominant disorder in which degeneration of medium‐sized spiny striatal neurons occurs. The HD gene and the protein it encodes, huntingtin, have been identified but their functions remain unknown. Transgenic mouse models for HD have been developed and we examined responses of medium‐sized striatal neurons recorded in vitro to application of N‐methyl‐D‐aspartate (NMDA) in two of these. The first model (R6/2) expresses exon 1 of the human HD gene with approximately 150 CAG repeats. In the R6/2 an enhancement of currents induced by selective activation of NMDA receptors as well as an enhancement of intracellular Ca2+ flux occurred in both presymptomatic and symptomatic mice. These alterations appeared specific for the NMDA receptor because α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor‐mediated currents were reduced in symptomatic R6/2s. In R6/2 animals there were parallel increases in NMDA‐R1 and decreases in NMDA‐R2A/B subunit proteins as established by immunohistochemistry. The second model (YAC72) contains human genomic DNA spanning the full‐length gene and all its regulatory elements with 72 CAG repeats. The phenotypical expression of the disorder develops more gradually than in the R6/2. In YAC72 mice we found similar but less marked increases in responses of medium‐sized striatal neurons to NMDA. These findings indicate that alterations in NMDA receptor function may predispose striatal neurons to excitotoxic damage, leading to subsequent neuronal degeneration and underscore the functional importance of NMDA receptors in HD. J. Neurosci. Res. 66:525–539, 2001.

Morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia.

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 Ca2+ currents and influx when depolarized. When these cells were acutely dissociated, peak Ca2+ 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.

Dopamine D4 Receptor-Deficient Mice Display Cortical Hyperexcitability

The dopamine D4 receptor (D4R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D4R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D4R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D4R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D4R can function as an inhibitory modulator of glutamate activity in the FC.

Dopamine Selects Glutamatergic Inputs to Neostriatal Neurons

Glutamatergic synaptic potentials induced by micromolar concentrations of the potassium conductance blocker 4‐aminopyridine (4‐AP) were recorded intracellularly from rat neostriatal neurons in the presence of 10 μM bicuculline (BIC). These synaptic potentials originate from neostriatal cortical and thalamic afferents and were completely blocked by 10 μM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) plus 100 μM D‐2‐amino‐5‐phosphonovaleric acid (2‐APV). Their inter‐event time intervals could be fitted to exponential distributions, suggesting that they are induced randomly. Their amplitude distributions had most counts around 1 mV and fewer counts with values up to 5 mV. Since input resistance of the recorded neurons is about 40 MΩ, the amplitudes agree to quantal size measurements in mammalian central neurons. The action of a D2 agonist, quinpirole, was studied on the frequency of these events. Mean amplitude of synaptic potentials was preserved in the presence of 2–10 μM quinpirole, but the frequency of 4‐AP‐induced glutamatergic synaptic potentials was reduced in 35% of cases. The effect was blocked by the D2 antagonist sulpiride (10 μM). Input resistance, membrane potential, or firing threshold did not change during quinpirole effect, suggesting a presynaptic site of action for quinpirole in some but not all glutamatergic afferents that make contact on a single cell. The present experiments show that dopaminergic presynaptic modulation of glutamatergic transmission in the neostriatum does not affect all stimulated afferents, suggesting that it is selective towards some of them. This may control the quality and quantity of afferent flow upon neostriatal neurons. Synapse 25:185–195, 1997.

Activity‐dependent bidirectional regulation of GABAA receptor channels by the 5‐HT4 receptor‐mediated signalling in rat prefrontal cortical pyramidal neurons

Emerging evidence has implicated a potential role for 5‐HT4 receptors in cognition and anxiolysis. One of the main target structures of 5‐HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5‐HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5‐HT4 receptor agonists produced either an enhancement or a reduction of GABA‐evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co‐expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5‐HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5‐HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5‐HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.

Pediatric Cortical Dysplasia: Correlations between Neuroimaging, Electrophysiology and Location of Cytomegalic Neurons and Balloon Cells and Glutamate/GABA Synaptic Circuits

Seizures in cortical dysplasia (CD) could be from cytomegalic neurons and balloon cells acting as epileptic ‘pacemakers’, or abnormal neurotransmission. This study examined these hypotheses using in vitro electrophysiological techniques to determine intrinsic membrane properties and spontaneous glutamatergic and GABAergic synaptic activity for normal-pyramidal neurons, cytomegalic neurons and balloon cells from 67 neocortical sites originating from 43 CD patients (ages 0.2–14 years). Magnetic resonance imaging (MRI), 18fluoro-2-deoxyglucose positron emission tomography (FDG-PET) and electrocorticography graded cortical sample sites from least to worst CD abnormality. Results found that cytomegalic neurons and balloon cells were observed more frequently in areas of severe CD compared with mild or normal CD regions as assessed by FDG-PET/MRI. Cytomegalic neurons (but not balloon cells) correlated with the worst electrocorticography scores. Electrophysiological recordings demonstrated that cytomegalic and normal-pyramidal neurons displayed similar firing properties without intrinsic bursting. By contrast, balloon cells were electrically silent. Normal-pyramidal and cytomegalic neurons displayed decreased spontaneous glutamatergic synaptic activity in areas of severe FDG-PET/MRI abnormalities compared with normal regions, while GABAergic activity was unaltered. In CD, these findings indicate that cytomegalic neurons (but not balloon cells) might contribute to epileptogenesis, but are not likely to be ‘pacemaker’ cells capable of spontaneous paroxysmal depolarizations. Furthermore, there was more GABA relative to glutamate synaptic neurotransmission in areas of severe CD. Thus, in CD tissue alternate mechanisms of epileptogenesis should be considered, and we suggest that GABAergic synaptic circuits interacting with cytomegalic and normal-pyramidal neurons with immature receptor properties might contribute to seizure generation.

Physiological and Molecular Properties of AMPA/Kainate Receptors Expressed by Striatal Medium Spiny Neurons

The mechanisms by which glutamate shapes the activity of striatal medium spiny neurons are of fundamental importance to our understanding of normative and pathological striatal physiology. Non-N-Methyl-D-aspartate (non-NMDA) glutamate receptor expression and function were studied in medium spiny neurons with a combination of single cell RT-PCR, immunocytochemistry and whole-cell voltage-clamp techniques. Reverse transcription polymerase chain reaction analysis found that GluR2 mRNA appeared to be the most abundant and widely distributed AMPA receptor mRNA. GluR1 was also commonly detected. However, GluR3 mRNA was preferentially expressed by neurons coexpressing substance P and enkephalin and GluR4 mRNA was not detected in identified medium spiny neurons. All neuronal classes appeared to express GluR5 or GluR6 and/or GluR7 mRNA in addition to kainate (KA) subunit mRNA. Immunocytochemical studies confirmed the mRNA distributions and also revealed that GluR1 protein was largely restricted to dendritic spines. Although the mRNA and protein for both α-amino-3-hydroxy-5-methyl-ioxyzole-4-proprionic acid (AMPA) and KA class subunits was detected, the physiological response to glutamatergic ligands and the benzothiadizine cyclothiazide was characteristic of AMPA, not KA receptors. The AMPA receptor antagonist GYKI 52466 blocked the response to AMPA and all but a small transient component of the response to KA. The current-voltage relationship of the AMPA-evoked currents was relatively linear but Ca2+ fluorometry revealed that substantial changes in intracellular Ca2+ concentration accompanied exposure to either agonist. These results argue that somatodendritic non-NMDA glutamate receptors in medium spiny neurons are primarily GluR2-containing receptors of the AMPA class but that activation of these receptors as a group nevertheless results in a significant Ca2+ influx.

Neurons Recorded from Pediatric Epilepsy Surgery Patients with Cortical Dysplasia

Summary: Purpose: Cortical dysplasia (CD) is a common pathological substrate in patients with early‐onset childhood epilepsy. In CD tissue, little is known about the mechanisms responsible for cellular hyperexcitability. In this study, we report initial electrophysiological and morphological observations from normal and dysmorphic cells in pediatric CD patients.

Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction

Recent data suggest that diabetes is a risk factor for pulmonary hypertension. The aim of the present study was to analyze whether diabetes induces endothelial dysfunction in pulmonary arteries and the mechanisms involved. Male Sprague-Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg(-1) streptozotocin). After 6 wk, intrapulmonary arteries were mounted for isometric tension recording, and endothelial function was tested by the relaxant response to acetylcholine. Protein expression and localization were measured by Western blot and immunohistochemistry and superoxide production by dihydroethidium staining. Pulmonary arteries from diabetic rats showed impaired relaxant response to acetylcholine and reduced vasoconstrictor response to the nitric oxide (NO) synthase inhibitor L-NAME, whereas the response to nitroprusside and the expression of endothelial NO synthase remained unchanged. Endothelial dysfunction was reversed by addition of superoxide dismutase or the NADPH oxidase inhibitor apocynin. An increase in superoxide production and increased expression of the NADPH oxidase regulatory subunit p47(phox) were also found in pulmonary arteries from diabetic rats. In conclusion, the pulmonary circulation is a target for diabetes-induced endothelial dysfunction via enhanced NADPH oxidase-derived superoxide production.

Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the rat.

Infrared videomicroscopy and differential interference contrast optics were used to identify medium‐ and large‐sized neurons in striatal slices from young rats. Whole‐cell patch‐clamp recordings were obtained to compare membrane currents evoked by application of N‐methyl‐d‐aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large‐ than in medium‐sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium‐ than in large‐sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large‐sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium‐sized neurons but were relatively infrequent in large‐sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large‐ than in medium‐sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium‐ and large‐sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time‐dependent increase in somatic area in medium‐sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium‐ and large‐sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders.