Amphetamine alters evoked responses of nigral neurons in kittens and adult cats

The effects of i.p. amphetamine administration (5 mg/kg) on the evoked unitary responses of substantia nigra (SN) neurons to electrical stimulation of their afferents were tested in 4 kittens (3–27 days of age) and 4 adult cats. In adults, amphetamine had two major effects: (1) it blocked temporarily (15–30 min) all neuronal responses to caudate (Cd) and cortical (Cx) stimulation; neuronal responsiveness recovered by 75 min post-drug; and (2) after 15 min postdrug, Cd and Cx stimulation evoked initial excitatory responses that were almost never found predrug. The latencies of Cd-evoked excitations indicated the existence of a mono- or oligosynaptic excitatory strionigral pathway while latencies of Cx-evoked excitations suggested that corticonigral excitatory influences were mediated multisynaptically. In kittens, amphetamine also produced an initial blockade of Cd- and Cx-evoked responses. However, the sign of initial responses to Cd stimulation was not altered since excitations were found both before and after drug treatment. These results indicated that amphetamine reveals excitatory evoked responses of SN neurons to striatal and striatally-mediated inputs that are masked during the course of normal postnatal development. Drug-related alterations of afferent inputs to SN neurons may underlie amphetamine-induced shifts in spontaneous neuronal activity which have been reported frequently.     

Dopamine attenuates evoked inhibitory synaptic currents in central amygdala neurons

The central nucleus of the amygdala (CeA) plays a critical role in regulating the behavioral, autonomic and endocrine response to stress. Dopamine (DA) participates in mediating the stress response and DA release is enhanced in the CeA during stressful events. However, the electrophysiological effects of DA on CeA neurons have not yet been characterized. Therefore, the purpose of this study was to identify and characterize the effect of DA application on electrophysiological responses of CeA neurons in coronal brain sections of male Sprague-Dawley rats. We used whole-cell patch-clamp electrophysiological techniques to record evoked synaptic responses and to determine basic membrane properties of CeA neurons both before and after DA superfusion. DA (20-250 μM) did not significantly alter membrane conductance over the voltage range tested. However, DA significantly reduced the peak amplitude of evoked inhibitory synaptic currents in CeA neurons. Pretreatment with the D(2) receptor antagonist eticlopride failed to significantly block the inhibitory effects of DA. In contrast, pretreatment with the D(1) receptor antagonist SCH-23390 significantly reduced the effects of DA on evoked inhibitory neurotransmission in these neurons. Moreover, bath superfusion of the specific D(1) receptor agonist SKF-39393, but not the D(2) receptor agonist quinpirole, significantly reduced peak amplitude of evoked inhibitory synaptic events. DA reduced the frequency of miniature IPSCs without altering the amplitude, while having no effect on the amplitude of IPSCs elicited by pressure application of GABA. These results suggest that DA may modulate inhibitory synaptic transmission in CeA through D(1) receptor activation primarily by a presynaptic mechanism.     

Caudate unit activity and somal diameters in intact and nigral lesioned cats

In a search for morphofunctional relationships in the head of the caudate nucleus (CN), we recorded extracellular unit activity in intact cats and in cats that had received bilateral injections of 6-OHDA into the substantia nigra (SN) 30 days previously. Only units firing spontaneously and continuously for 2 min were studied. In dorsal regions, potentials were small and iterative at almost constant intervals; the somal diameters were relatively small. In the ventrolateral region, potentials were bigger and appeared in bursts; somal diameters were significantly larger (p less than 0.05). For the centromedial region a histogram of numbers of neurons as a function of diameters revealed a Gaussian distribution extending from small to large neurons. Most dorsal neurons increased their firing rate to radial nerve, visual, SN, and/or nucleus centralis medialis (NCM) stimulation. Ventral neurons usually responded with excitation followed by long lasting inhibition, particularly to SN and NCM stimulation. A few neurons responded to all four inputs and some showed long-lasting potentiation in response to low frequency stimulation, suggesting a more general function. Greatest convergence (65%) was found for NCM and SN inputs. In lesioned cats, there was no SN driving, NCM's inhibitory actions almost disappeared, and the excitatory action of the other stimuli was reduced.     

Caudate neurons: Cholinoceptive receptor sites

The effects of acetylcholine, atropine, dopamine, γ-aminobutyric acid, glutamate, serotonin, and tubocurarine microiontophoretic pulses on unitary activity and locally evoked field potentials in the chronically isolated caudate nucleus of rats and cats were studied. Acetylcholine emerged as the most effective ion capable of influencing the unitary and neuronal pool activities of the tissue at the recording pore, overwhelmingly in the direction of excitation of silent units and enhancement of focal potentials. Atropine failed to block these excitatory effects but tubocurarine was found to suppress ACh-induced firing in some units. On the basis of this and other electrophysiologic and morphologic evidence we conclude that the few spontaneously firing and a substantial representation of silent neurons of the caudate nucleus are cholinoceptive and arranged in closely packed and interconnected clusters such that focal microstimulations, neurochemical and electrical, are capable of inducing their firing singly or as a neuronal population potential. The overwhelming cholinoceptive properties of neurons in isolated caudates corroborate the lesion and biochemical data indicating that acetylcholine, its precursor, and their related enzymes are harbored within intrinsic elements of the caudate nucleus.     

Characteristics of spontaneous and evoked GABAergic synaptic currents in cardiac vagal neurons in rats

Despite the importance of GABAergic input to cardiac vagal neurons the electrophysiological properties and possible origins of this innervation have not yet been studied. Individual cardiac vagal neurons were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Cardiac vagal neurons received spontaneous GABAergic inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA(A) receptor antagonist bicuculline. The spontaneous presynaptic GABAergic input to cardiac vagal neurons in the nucleus ambiguus occurred at a significantly lower frequency than that recorded in cardiac vagal neurons in the dorsal motor nucleus of the vagus. To identify a possible source of the GABAergic innervation to cardiac vagal neurons the nucleus tractus solitarius was electrically stimulated. GABAergic synaptic currents in cardiac vagal neurons, in both the dorsal motor nucleus of the vagus (DMNX) and the nucleus ambiguus (NA), were consistently evoked upon stimulation of the nucleus tractus solitarius and these responses were also blocked by bicuculline.     

Altered patterns of evoked synaptic activity in cortical pyramidal neurons in feline ganglioside storage disease

Postsynaptic potentials evoked by ventrolateral thalamic stimulation were recorded intracellularly from neurons in the precruciate cortex of GM1 mutants with HRP- or LY-loaded microelectrodes. Ganglioside-laden pyramidal neurons exhibiting somal distention and/or meganeurite formation were found to respond to thalamic stimulation with short duration IPSPs. Evoked EPSPs were recorded from two morphologically characterized large basket intrinsic neurons which deployed extensive intracortical axonal arborizations. These findings point to the preservation of intracortical inhibitory networks in the feline model of GM1 gangliosidosis, and to the possibility of abnormal integration of somadendritic inputs in ganglioside-laden pyramidal neurons.     

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission

Glial cells express specific high-affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole-cell patch-clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium-dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.     

Correlation of miniature synaptic activity and evoked release probability in cultures of cortical neurons.

Spontaneous miniature synaptic activity is caused by action potential (AP)-independent release of transmitter vesicles and is regulated at the level of single synapses. In cultured cortical neurons we have used this spontaneous vesicle turnover to load the styryl dye FM1-43 into synapses with high rates of miniature synaptic activity. Automated selection procedures restricted analysis to synapses with sufficient levels of miniature activity-mediated FM1-43 uptake. After FM1-43 loading, vesicular FM1-43 release in response to AP stimulation was recorded at single synapses as a measure of release probability. We find that synapses with high rates of miniature activity possess significantly enhanced evoked release rates compared with a control population. Because the difference in release rates between the two populations is [Ca(2+)](o)-dependent, it is most likely caused by a difference in release probability. Within the subpopulation of synapses with high miniature activity, we find that the probabilities for miniature and AP-evoked release are correlated at single synaptic sites. Furthermore, the degree of miniature synaptic activity is correlated with the vesicle pool size. These findings suggest that both evoked and miniature vesicular release are regulated in parallel and that the frequency of miniature synaptic activity can be used as an indicator for evoked release efficacy.     

Nifedipine blocks apamin-induced bursting activity in nigral dopamine-containing neurons

Intrinsic sinusoidal oscillations in membrane potential, characteristic of nigral dopamine cells, are converted to plateau potentials following application of apamin, a potent antagonist of SK-type Ca²⁺-activated K⁺ channels. Blockade of these channels also changes neuronal firing pattern from a single-spike pacemaker discharge to a multiple spike bursting pattern. Nifedipine, a selective antagonist of L-type Ca²⁺ channels, blocks plateau potential generation; however, its effects on firing pattern have yet to be determined. In the present study, extracellular single unit recording techniques were used in conjunction with a brain slice preparation to determine whether nifedipine, in a concentration known to block plateau potential generation, also affects bursting activity. Nifedipine (30 μM) was equipotent in inhibiting the firing rate of control (51.2±10.8%) and apamin-treated (44.9±5.4%) neurons. Slow firing neurons (<2 Hz) were particularly sensitive to the inhibitory effects of the drug. Apamin-induced bursting was completely suppressed by nifedipine and accompanied by a significant increase in the regularity of firing. By contrast, pacemaker-like activity exhibited by control neurons was unaffected by the drug. These data demonstrate that the intrinsic plateau properties exhibited by DA neurons are responsible for the generation of phasic activity induced following blockade of apamin-sensitive Ca²⁺-activated K⁺ channels and provide further support for the involvement of an L-type Ca²⁺ conductance in mediating this type of activity.     

Spontaneous synaptic activities in rat nucleus tractus solitarius neurons in vitro: evidence for re-excitatory processing

The pattern of synaptic interactions between neurons of the nucleus tractus solitarius (NTS) has been analyzed using whole cell recording in rat brainstem slices. Following tractus solitarius (TS) stimulation 15/55 neurons presented a prolonged (up to 300 ms) increased excitability (PIE neurons) and 40/55 neurons presented a prolonged (up to 200 ms) reduced excitability (PRE neurons). In the absence of afferent sensory input all neurons showed spontaneous synaptic activity. Onggoing synaptic activity in PIE cells was glutamatergic and characterized by the absence of detectable inhibitory potentials while in PRE cells it was 90% GABAergic and 10% glutamatergic. Glutaminergic synaptic currents in PIE cells and GABAergic synaptic currents in PRE were studied using probability density and intensity functions. Distribution of time intervals between synaptic events indicated the latter were generated, in both PIE and PRE cells, by two simultaneous processes: (1) a close to Poisson process generating independent events; and (2) a subsidiary re-excitatory process generating synaptic events separated by intervals shorter than 20 ms. Blockade of glutamatergic transmission by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) or blockade of action potentials by tetrodotoxin (TTX; 1 μM) suppressed the subsidiary process. In conclusion, we propose that PIE cells (1) form a re-excitatory network contributing to generation of excitatory activity in the NTS and (2) are located presynaptically with respect to PRE cells.     

Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons

Dysfunction of the ubiquitin-proteasomal system (UPS) has been implicated in the pathogenesis of Parkinson's disease. The systemic administration of UPS inhibitors has been reported to induce nigrostriatal cell death and model Parkinson's disease pathology in rodents. We administered a synthetic, specific UPS inhibitor (PSI) subcutaneously to rats and quantified substantia nigral tyrosine hydroxylase-positive dopaminergic neurons by stereology. PSI caused a 15% decrease in UPS activity at 2 weeks and a 42% reduction in substantia nigra pars compacta tyrosine hydroxylase-positive neurons at 8 weeks. Systemic inhibition of the UPS warrants further evaluation as a means to model Parkinson's disease.     

MPP(+) and glutamate in the degeneration of nigral dopaminergic neurons

Glutamate neurotoxicity can be an experimental oxidative stress, and we investigated glutamate toxicity against cultured rat mesencephalic neurons. Although glutamate showed similar toxicity against dopaminergic and nondopaminergic neurons, nitric oxide (NO) showed neurotoxicity restricted exclusively in nondopaminergic neurons. An inhibitor of NO synthase had no significant effect on the glutamate toxicity against dopaminergic neurons, however, it had a significant antagonistic effect on that against nondopaminergic neurons. These findings indicate the presence of two mechanisms of glutamate neurotoxicity, one being not mediated by NO, found in dopaminergic neurons, and the other being mediated via NO, found in nondopaminergic neurons. In contrast to NO, peroxynitrite (ONOO⁻), an active metabolite of NO, caused significant cytotoxicity against dopaminergic and nondopaminergic neurons, suggesting that conversion of NO to ONOO⁻ is suppressed in dopaminergic neurons. After pretreatment with small doses of methyl-4-phenylpyridium ion (MPP⁺), NO caused significant cytotoxicity against dopaminergic neurons, and glutamate toxicity was enhanced only against dopaminergic neurons. Therefore, sublethal dose of MPP⁺ enhances glutamate toxicity against dopaminergic neurons, probably by the facilitation of suppressed NO conversion to ONOO⁻ in dopaminergic neurons. Finally, to provide basic data for neuroprotective therapy in Parkinson's disease, we investigated neuroprotection against glutamate toxicity by dopamine agonists. Preincubation with the D2 type dopamine agonists provides neuroprotection against glutamate neurotoxicity and the protective effects blocked by a D2 antagonist, indicating that D2 agonists provide protection mediated not only by the inhibition of dopamine turnover, but also via D2 type dopamine receptor.     

Two types of spheroid bodies in the nigral neurons in Parkinson's disease

Dendritic spheroid bodies (SBs) and Lewy bodies (LBs) were identified in comparable numbers in the substantia nigra pars compacta (SBC) of nine parkinsonian cases and one case of striatonigral degeneration but were not found in cases of Huntington's disease or neurologically normal controls. The immunohistochemical profile of the SBs in dystrophic dendrites of nigrostriatal dopaminergic neurons was remarkably similar to that of the LBs found within dendrites or free of the SNC neuropil. Both types of inclusions stained positively with antibodies to tyrosine hydroxylase, ubiquitin and microtubule-associated protein-2 (MAP2), and negatively for Tau-2, although they had different ultrastructural appearances. A few intracellular LBs were stained by antibodies to neurofilament proteins (NFs) 68, 160, and 200 kD, but dendritic SBs and extracellular LBs were not so stained. These data indicate that dendritic SBs and extracellular LBs may have a common molecular pathogenetic origin in Parkinson's disease. On the other hand, the SBs seen in the pars reticulata (SNR) and in the distal nigrostriatal axons even in control cases were generally stained by antibodies to NFs and ubiquitin but not to MAP2. This latter staining pattern in similar to that shown by SBs in the anterior horn in ALS and in the cerebellum of neurologically normal brains and is believed typical of axonal as opposed to dendritic SBs.     

Caspase inhibition protects nigral neurons against 6-OHDA-induced retrograde degeneration

6-Hydroxydopamine (6-OHDA) administered intrastriatally to adult rats in a single injection causes neurodegeneration of the nigrostriatal pathway and loss of > 50% of dopamine neurons in substantia nigra pars compacta 30 days after administration. The death of nigral neurons occurs, at least partially, by a caspase-mediated mechanism. The nigral loss of dopaminergic neurons could be prevented by stereotaxical administration of zVAD.fmk, a caspase inhibitor, into the substantia nigra, indicating that 6-OHDA-induced nigrostriatal degeneration involves caspase activation. These results suggest that caspases are probably involved in neurodegenerative chronic processes such as Parkinson's disease and might be considered as possible targets in the treatment of such neurological disorders.     

Evidence of deprenyl-insensitive apoptosis of nigral dopamine neurons during development

Apoptosis of dopamine neurons occurs naturally in the substantia nigra during development, culminating in approximately 30% loss of these cells during the perinatal period. Deprenyl, independent of its monoamine oxidase (MAO)-B inhibitory properties, can prevent dopamine neuronal apoptosis in models of neurodegeneration. Our current study demonstrate that apoptotic death of dopamine neurons during development is insensitive to daily treatment of pregnant mothers and then newborns with deprenyl (0.1, 1, or 10 mg/kg). This result is not due to poor crossing of the placental and blood-brain barriers, since deprenyl caused a dose-dependent inhibition of brain MAO-B activity in pups at birth. Determining the pathway(s) leading to deprenyl-insensitive apoptosis of nigral dopamine neurons in development may shed light on mechanisms underlying the premature death of dopamine neurons in neurodegenerative disorders.     

Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinson's disease models

Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic (DA) neuronal cell loss in the substantia nigra. Although the entire pathogenesis of PD is still unclear, both environmental and genetic factors contribute to neurodegeneration. Epidemiologic studies show that prevalence of PD is lower in smokers than in nonsmokers. Nicotine, a releaser of dopamine from DA neurons, is one of the candidates of antiparkinson agents in tobacco. To assess the protective effect of nicotine against rotenone-induced DA neuronal cell toxicity, we examined the neuroprotective effects of nicotine in rotenone-induced PD models in vivo and in vitro. We observed that simultaneous subcutaneous administration of nicotine inhibited both motor deficits and DA neuronal cell loss in the substantia nigra of rotenone-treated mice. Next, we analyzed the molecular mechanisms of DA neuroprotective effect of nicotine against rotenone-induced toxicity with primary DA neuronal culture. We found that DA neuroprotective effects of nicotine were inhibited by dihydro-beta-erythroidine (DHbetaE), alpha-bungarotoxin (alphaBuTx), and/or PI3K-Akt/PKB (protein serine/threonine kinase B) inhibitors, demonstrating that rotenone-toxicity on DA neurons are inhibited via activation of alpha4beta2 or alpha7 nAChRs-PI3K-Akt/PKB pathway or pathways. These results suggest that the rotenone mouse model may be useful for assessing candidate antiparkinson agents, and that nAChR (nicotinic acetylcholine receptor) stimulation can protect DA neurons against degeneration.     

The control of firing pattern in nigral dopamine neurons: burst firing

In addition to firing in a single spiking mode, dopamine (DA) cells have been observed to fire in a bursting pattern with consecutive spikes in a burst displaying progressively decreasing amplitude and increasing duration. In vivo intracellular recording demonstrated the bursts to typically ride on a depolarizing wave (5 to 15 mV amplitude). Although the burst-firing frequency of DA cells showed little correlation with the base line firing rate, increases in firing rate were usually associated with an increase in burst firing. Increases in burst firing could also be elicited by intracellular calcium injection and could be prevented by intracellular injection of EGTA, suggesting a calcium involvement in bursting. Blockade of potassium conductances with extracellular iontophoresis of barium or intracellular injection of tetraethylammonium bromide could also trigger an increased degree of burst firing in DA cells. These data suggest that the increased calcium influx accompanying an increased firing rate triggers burst firing, possibly by inactivating a potassium conductance. A switch from a single spiking mode to a burst-firing mode may be important in modulating striatal DA release, as shown for burst firing in other preparations.