Paolo Gubellini

Acetylcholine-mediated modulation of striatal function

Striatal spiny neurones serve as a major anatomical locus for the relay of cortical information flow through the basal ganglia. these projection neurones also represent the main synaptic target of cholinergic interneurones, whose physiological role in striatal activity still remains largely enigmatic. The striatal cholinergic system has been implicated in the pathophysiology of movement disorders such as Parkinsons disease, but the cellular mechanisms underlying cholinergic-neurone function are still unknown. On the basis of in vitro electrophysiological evidence, obtained from a rat corticostriatal-slice preparation, we propose that endogenous ACh exerts a complex modulation of striatal synaptic transmission, which produces both short-term and long-term effects. ACh-mediated mechanisms might be of crucial importance in processing the cortical inputs to the striatum.

Dopaminergic control of synaptic plasticity in the dorsal striatum

Cortical glutamatergic and nigral dopaminergic afferents impinge on projection spiny neurons of the striatum, providing the most significant inputs to this structure. Isolated activation of glutamate or dopamine (DA) receptors produces short‐term effects on striatal neurons, whereas the combined stimulation of both glutamate and DA receptors is able to induce long‐lasting modifications of synaptic excitability. Repetitive stimulation of corticostriatal fibres causes a massive release of both glutamate and DA in the striatum and, depending on the glutamate receptor subtype preferentially activated, produces either long‐term depression (LTD) or long‐term potentiation (LTP) of excitatory synaptic transmission. D1‐like and D2‐like DA receptors interact synergistically to allow LTD formation, while they operate in opposition during the induction phase of LTP. Corticostriatal synaptic plasticity is severely impaired after chronic DA denervation and requires the stimulation of DARPP‐32, a small protein expressed in dopaminoceptive spiny neurons which acts as a potent inhibitor of protein phosphatase‐1. In addition, the formation of LTD and LTP requires the activation of PKG and PKA, respectively, in striatal projection neurons. These kinases appear to be stimulated by the activation of D1‐like receptors in distinct neuronal populations.

Experimental Parkinsonism Alters Endocannabinoid Degradation: Implications for Striatal Glutamatergic Transmission

Cannabinoid receptors and their endogenous ligands have been recently identified in the brain as potent inhibitors of neurotransmitter release. Here we show that, in a rat model of Parkinsons disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of anandamide, but not that of the other endocannabinoid 2-arachidonoylglycerol, were increased. Moreover, we observed a decreased activity of the anandamide membrane transporter (AMT) and of the anandamide hydrolase [fatty acid amide hydrolase (FAAH)], whereas the binding of anandamide to cannabinoid receptors was unaffected. Spontaneous glutamatergic activity recorded from striatal spiny neurons was higher in 6-OHDA-lesioned rats. Inhibition of AMT byN-(4-hydroxyphenyl)-arachidonoylamide (AM-404) or by VDM11, or stimulation of the cannabinoid CB1 receptor by HU-210 reduced glutamatergic spontaneous activity in both naı̈ve and 6-OHDA-lesioned animals to a similar extent. Conversely, the FAAH inhibitors phenylmethylsulfonyl fluoride and methyl-arachidonoyl fluorophosphonate were much more effective in 6-OHDA-lesioned animals. The present study shows that inhibition of anandamide hydrolysis might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinsons disease.

Synaptic transmission in the striatum: from plasticity to neurodegeneration

Striatal neurones receive myriad of synaptic inputs originating from different sources. Massive afferents from all areas of the cortex and the thalamus represent the most important source of excitatory amino acids, whereas the nigrostriatal pathway and intrinsic circuits provide the striatum with dopamine, acetylcholine, GABA, nitric oxide and adenosine. All these neurotransmitter systems interact each other and with voltage-dependent conductances to regulate the efficacy of the synaptic transmission within this nucleus. The integrative action exerted by striatal projection neurones on this converging information dictates the final output of the striatum to the other basal ganglia structures. Recent morphological, immunohistochemical and electrophysiological findings demonstrated that the striatum also contains different interneurones, whose role in physiological and pathological conditions represents an intriguing challenge in these years. The use of the in vitro brain slice preparation has allowed not only the detailed investigation of the direct pre- and postsynaptic electrophysiological actions of several neurotransmitters in striatal neurones, but also the understanding of their role in two different forms of corticostriatal synaptic plasticity, long-term depression and long-term potentiation. These long-lasting changes in the efficacy of excitatory transmission have been proposed to represent the cellular basis of some forms of motor learning and are altered in animal models of human basal ganglia disorders, such as Parkinsons disease. The striatum also expresses high sensitivity to hypoxic-aglycemic insults. During these pathological conditions, striatal synaptic transmission is altered depending on presynaptic inhibition of transmitter release and opposite membrane potential changes occur in projection neurones and in cholinergic interneurones. These ionic mechanisms might partially explain the selective neuronal vulnerability observed in the striatum during global ischemia and Huntingtons disease.

Receptor subtypes involved in the presynaptic and postsynaptic actions of dopamine on striatal interneurons

By stimulating distinct receptor subtypes, dopamine (DA) exerts presynaptic and postsynaptic actions on both large aspiny (LA) cholinergic and fast-spiking (FS) parvalbumin-positive interneurons of the striatum. Lack of receptor- and isoform-specific pharmacological agents, however, has hampered the progress toward a detailed identification of the specific DA receptors involved in these actions. To overcome this issue, in the present study we used four different mutant mice in which the expression of specific DA receptors was ablated. In D1 receptor null mice, D1R-/-, DA dose-dependently depolarized both LA and FS interneurons. Interestingly, SCH 233390 (10 μm), a D1-like (D1 and D5) receptor antagonist, but not l-sulpiride (3–10 μm), a D2-like (D2, D3, D4) receptor blocker, prevented this effect, implying D5 receptors in this action. Accordingly, immunohistochemical analyses in both wild-type and D1R-/- mice confirmed the expression of D5 receptors in both cholinergic and parvalbumin-positive interneurons of the striatum. In mice lacking D2 receptors, D2R-/-, the DA-dependent inhibition of GABA transmission was lost in both interneuron populations. Both isoforms of D2 receptor, D2L and D2S, were very likely involved in this inhibitory action, as revealed by the electrophysiological analysis of the effect of the DA D2-like receptor agonist quinpirole in two distinct mutants lacking D2L receptors and expressing variable contents of D2S receptors. The identification of the receptor subtypes involved in the actions of DA on different populations of striatal cells is essential to understand the circuitry of the basal ganglia and to develop pharmacological strategies able to interfere selectively with specific neuronal functions.

Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory

Induction of long‐term depression (LTD) in rat striatal slices revealed that this form of synaptic plasticity is coupled to an increased expression of tissue‐plasminogen activator (t‐PA) mRNA, as detected by the mRNA differential display technique. To further investigate the involvement of this gene in synaptic remodelling following striatal LTD, we recorded electrical activity from mice lacking the gene encoding t‐PA (t‐PA‐KO) and from wild‐type (WT) mice. Tetanic stimulation induced LTD in the large majority of striatal neurons recorded from WT mice. Conversely, LTD was absent in a significant proportion of striatal neurons obtained from mice lacking t‐PA. Electrophysiological recordings obtained from hippocampal slices in the CA1 area showed that mainly the late phase of long‐term potentiation (LTP) was reduced in t‐PA‐KO mice. Learning and memory‐related behavioural abnormalities were also found in these transgenic mice. Disruption of the t‐PA gene, in fact, altered both the context conditioning test, a hippocampus‐related behavioural task, and the two‐way active avoidance, a striatum‐dependent task. In an open field object exploration task, t‐PA‐KO mice expressed deficits in habituation and reactivity to spatial change that are consistent with an altered hippocampal function. Nevertheless, decreased rearing and poor initial object exploration were also observed, further suggesting an altered striatal function. These data indicate that t‐PA plays a critical role in the formation of various forms of synaptic plasticity and memory.

ENDOGENOUS ACH ENHANCES STRIATAL NMDA-RESPONSES VIA M1-LIKE MUSCARINIC RECEPTORS AND PKC ACTIVATION

Cortical glutamatergic fibres and cholinergic inputs arising from large aspiny interneurons converge on striatal spiny neurons and play a major role in the control of motor activity. We have investigated the interaction between excitatory amino acids and acetylcholine (ACh) on striatal spiny neurons by utilizing intracellular recordings, both in current‐ and in voltage‐clamp mode in rat brain slices. Muscarine (0.3–10 μm) produced a reversible and dose‐dependent increase in the membrane depolarizations/inward currents induced by brief applications of N‐methyl‐d‐aspartate (NMDA), while it did not affect the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)‐induced responses. These concentrations of muscarine did not alter the membrane potential and the current‐voltage relationship of the recorded cells. Neostigmine (0.3–10 μm), an ACh‐esterase inhibitor, mimicked this facilitatory effect. The facilitatory effects of muscarine and neostigmine were antagonized either by scopolamine (3 μm) or by pirenzepine (10–100 nm), an antagonist of M1‐like muscarinic receptors, but not by methoctramine (300 nm), an antagonist of M2‐like muscarinic receptor. Accordingly, these facilitatory effects were mimicked by McN‐A‐343 (1–10 μm), an agonist of M1‐like muscarinic receptors, but not by oxotremorine (300 nm), an agonist of M2‐like receptors. Tetrodotoxin (TTX) did not block the facilitatory effect produced by the activation of muscarinic receptors suggesting that this effect is postsynaptically mediated. The action of neostigmine was prevented either by the intracellular calcium (Ca2+) chelator BAPTA (200 mm) or by preincubating the slices with inhibitors of protein kinase C (PKC) (staurosporine 100 nm or calphostin C 1 μm). McN‐A‐343 did not alter the excitatory post synaptic potentials (EPSPs) evoked by corticostriatal stimulation in the presence of physiological concentration of magnesium (Mg2+ 1.2 mm), while it enhanced the duration of these EPSPs recorded in the absence of external magnesium. Our data show that endogenous striatal ACh exerts a positive modulatory action on NMDA responses via M1‐like muscarinic receptors and PKC activation.

Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonism

Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinsons disease induced by unilateral nigral lesion with 6‐hydroxydopamine (6‐OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty‐acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate‐mediated spontaneous excitatory post‐synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6‐OHDA‐lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinsons disease.

Dopamine, acetylcholine and nitric oxide systems interact to induce corticostriatal synaptic plasticity.

Two distinct forms of synaptic plasticity have been described at corticostriatal synapses: long-term depression (LTD) and long-term potentiation (LTP). Both these enduring changes in the efficacy of excitatory neurotransmission in the striatum have a major impact on the physiological activity of the basal ganglia and are triggered by the stimulation of complex and independent cascades of intracellular second messenger systems. Along with the massive glutamatergic inputs originating from the cortex, striatal neurons receive a myriad of other synaptic contacts arising from different sources. In particular, while the nigrostriatal pathway provides this brain area with dopamine (DA), intrinsic circuits are the main source of acetylcholine (ACh) and nitric oxide (NO). The three neurotransmitter systems interact with each other to determine whether corticostriatal LTP or LTD is triggered in response to repetitive synaptic stimulation. Two distinct subtypes of striatal interneurons produce ACh and NO in the striatum. These interneurons are activated by the cortex during the induction phase of striatal plasticity, and stimulate, in turn, the intracellular changes in projection neurons required for LTD or LTP. Interneurons, therefore, exert a feedforward control of the excitability of striatal projection neurons by ensuring the coordinate expression of two alternative forms of synaptic plasticity at the same type of excitatory synapse. The integrative action exerted by striatal projection neurons on the converging information arising from the cortex, nigral DA neurons, and from ACh- and NO-producing interneurons dictates the final output of the striatum to the other structures of the basal ganglia.

Deep brain stimulation in neurological diseases and experimental models: from molecule to complex behavior.

Deep brain stimulation (DBS) has proven to be capable of providing significant benefits for several neuropathologies. It is highly effective in reducing the motor symptoms of Parkinsons disease, essential tremor, and dystonia, and in alleviating chronic pain. Recently, also Tourette syndrome, obsessive-compulsive disorder and treatment-resistant depression have been treated by DBS with encouraging results. However, despite these clinical achievements, the precise action mechanisms of DBS still need to be fully characterized. For this reason, several animal models of DBS have been developed, bringing new insights on the effects of this treatment at molecular and cellular level, and providing new evidence on its physiological and behavioral consequences. In parallel, physiological and imaging studies in patients have contributed to better understanding DBS impact on the function of brain circuits. Here we review the clinical data and experimental work in vitro, ex vivo and in vivo (mostly arisen from studies on DBS of the subthalamic nucleus) in the treatment of PD, which led to the actual knowledge of DBS mechanisms, from molecular to complex behavioral levels.