Craig P. Blomeley

Excitatory effects of serotonin on rat striatal cholinergic interneurones

We investigated the effects of 5‐hydroxytryptamine (5‐HT, serotonin) in striatal cholinergic interneurones with gramicidin‐perforated whole‐cell patch recordings. Bath‐application of serotonin (30 μm) significantly and reversibly increased the spontaneous firing rate of 37/45 cholinergic interneurones tested. On average, in the presence of serotonin, firing rate was 273 ± 193% of control. Selective agonists of 5‐HT1A, 5‐HT3, 5‐HT4 and 5‐HT7 receptors did not affect cholinergic interneurone firing, while the 5‐HT2 receptor agonist α‐methyl‐5‐HT (30 μm) mimicked the excitatory effects of serotonin. Consistently, the 5‐HT2 receptor antagonist ketanserin (10 μm) fully blocked the excitatory effects of serotonin. Two prominent after‐hyperpolarizations (AHPs), one of medium duration that was apamin‐sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization‐activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5‐HT, and fully abolished by the calcium channel blocker nickel. These results show that 5‐HT exerts a powerful excitatory control on cholinergic interneurones via 5‐HT2 receptors, by suppressing the AHPs associated with two distinct calcium‐activated potassium currents.

Serotonin excites fast-spiking interneurons in the striatum

Fast‐spiking interneurons (FSIs) control the output of the striatum by mediating feed‐forward GABAergic inhibition of projection neurons. Their neuromodulation can therefore critically affect the operation of the basal ganglia. We studied the effects of 5‐hydroxytryptamine (5‐HT, serotonin), a neurotransmitter released in the striatum by fibres originating in the raphe nuclei, on FSIs recorded with whole‐cell techniques in rat brain slices. Bath application of serotonin (30 μm) elicited slow, reversible depolarizations (9 ± 3 mV) in 37/46 FSIs. Similar effects were observed using conventional whole‐cell and gramicidin perforated‐patch techniques. The serotonin effects persisted in the presence of tetrodotoxin and were mediated by 5‐HT2C receptors, as they were reversed by the 5‐HT2 receptor antagonist ketanserin and by the selective 5‐HT2C receptor antagonist RS 102221. Serotonin‐induced depolarizations were not accompanied by a significant change in FSI input resistance. Serotonin caused the appearance of spontaneous firing in a minority (5/35) of responsive FSIs, whereas it strongly increased FSI excitability in each of the remaining responsive FSIs, significantly decreasing the latency of the first spike evoked by a current step and increasing spike frequency. Voltage‐clamp experiments revealed that serotonin suppressed a current that reversed around −100 mV and displayed a marked inward rectification, a finding that explains the lack of effects of serotonin on input resistance. Consistently, the effects of serotonin were completely occluded by low concentrations of extracellular barium, which selectively blocks Kir2 channels. We concluded that the excitatory effects of serotonin on FSIs were mediated by 5‐HT2C receptors and involved suppression of an inwardly rectifying K+ current.

Substance P mediates excitatory interactions between striatal projection neurons.

The striatum is the largest nucleus of the basal ganglia, and is crucially involved in motor control. Striatal projection cells are medium-size spiny neurons (MSNs) and form functional GABAergic synapses with other MSNs through their axon collaterals. A subpopulation of MSNs also release substance P (SP), but its role in MSN–MSN communication is unknown. We studied this issue in rat brain slices, in the presence of antagonists for GABA, acetylcholine, dopamine, and opioid receptors; under these conditions, whole-cell paired recordings from MSNs (located <100 μm apart) revealed that, in 31/137 (23%) pairs, a burst of five spikes in a MSN caused significant facilitation (14.2 ± 8.9%) of evoked glutamatergic responses in the other MSN. Reciprocal facilitation of glutamatergic responses was present in 4 of these pairs. These facilitatory effects were maximal when spikes preceded glutamatergic responses by 100 ms, and were completely blocked by the NK1 receptor antagonist L-732,138. Furthermore, in 31/57 (54%) MSNs, a burst of 5 antidromic stimuli delivered to MSN axons in the globus pallidus significantly potentiated glutamatergic responses evoked 250 or 500 ms later by stimulation of the corpus callosum. These effects were larger at 250 than 500 ms intervals, were completely blocked by L-732,138, and facilitated spike generation. These data demonstrate that MSNs facilitate glutamatergic inputs to neighboring MSNs through spike-released SP acting on NK1 receptors. The current view that MSNs form inhibitory networks characterized by competitive dynamics will have to be updated to incorporate the fact that groups of MSNs interact in an excitatory manner.

Opioidergic Interactions between Striatal Projection Neurons

Medium spiny striatal projection neurons (MSNs) release opioid neuropeptides, but the role of these neurotransmitters is still poorly understood. While presynaptic inhibition of corticostriatal axons by opioid receptors has been demonstrated using exogenous ligands, the action of synaptically released opioids in the striatum has not been investigated. We performed single and paired whole-cell recordings from rat MSNs while corticostriatal fibers were electrically activated. In single recording experiments, we also activated antidromically the axons of a population of MSNs. Corticostriatal fibers were stimulated once every 10 s and every other stimulation was preceded by 5 antidromic spikes (at 100 Hz). This burst of antidromic spikes produced robust inhibition of evoked corticostriatal responses. This inhibition was not affected by the δ-opioid receptor antagonist SDM25N, but was completely abolished by the μ-opioid receptor antagonist CTOP. Inhibitory effects were maximal (on average 29.6 ± 11.4%) when the burst preceded the corticostriatal stimulation by 500 ms and became undetectable for intervals >2 s. Paired recordings from MSNs located <100 μm apart revealed that, in 30 of 56 (54%) pairs, a burst of five action potentials in one of the MSNs caused significant inhibition (17.1 ± 5.7%) of evoked glutamatergic responses in the other MSN. In 5 of these pairs, reciprocal inhibition of corticostriatal inputs was present. These effects were maximal 500 ms after the burst and were completely blocked by CTOP. Thus, these results reveal a novel, strong opioid-mediated communication between MSNs and provide a new cellular substrate for competitive dynamics in the striatum.

Ethanol Affects Striatal Interneurons Directly and Projection Neurons Through a Reduction in Cholinergic Tone

The acute effects of ethanol on the neurons of the striatum, a basal ganglia nucleus crucially involved in motor control and action selection, were investigated using whole-cell recordings. An intoxicating concentration of ethanol (50 mM) produced inhibitory effects on striatal large aspiny cholinergic interneurons (LAIs) and low-threshold spike interneurons (LTSIs). These effects persisted in the presence of tetrodotoxin and were because of an increase in potassium currents, including those responsible for medium and slow afterhyperpolarizations. In contrast, fast-spiking interneurons (FSIs) were directly excited by ethanol, which depolarized these neurons through the suppression of potassium currents. Medium spiny neurons (MSNs) became hyperpolarized in the presence of ethanol, but this effect did not persist in the presence of tetrodotoxin and was mimicked and occluded by application of the M1 muscarinic receptor antagonist telenzepine. Ethanol effects on MSNs were also abolished by 100 μM barium. This showed that the hyperpolarizations observed in MSNs were because of decreased tonic activation of M1 muscarinic receptors, resulting in an increase in Kir2 conductances. Evoked GABAergic responses of MSNs were reversibly decreased by ethanol with no change in paired-pulse ratio. Furthermore, ethanol impaired the ability of thalamostriatal inputs to inhibit a subsequent corticostriatal glutamatergic response in MSNs. These results offer the first comprehensive description of the highly cell type-specific effects of ethanol on striatal neurons and provide a cellular basis for the interpretation of ethanol influence on a brain area crucially involved in the motor and decisional impairment caused by this drug.

Substance P depolarizes striatal projection neurons and facilitates their glutamatergic inputs.

The striatum is the main basal ganglia input nucleus, receiving extensive glutamatergic inputs from cortex and thalamus. Medium spiny striatal projection neurons (MSNs) are GABAergic, and their axon collaterals synapse on other MSNs. Approximately 50% of MSNs corelease substance P (SP), but how this neurotransmitter controls MSN activity is poorly understood. We used whole‐cell recordings to investigate how SP affects MSNs and their glutamatergic inputs. SP elicited slow depolarizations in 47/90 MSNs, which persisted in the presence of tetrodotoxin (TTX). SP responses were mimicked by the NK1 receptor agonist [Sar9,Met(O2)11]‐substance P (SSP), and fully blocked by the NK1 receptor antagonists L‐732,138, or extracellular zinc. When intracellular chloride was altered, the polarity of SP responses depended on the sign of the chloride driving force. In voltage‐clamp, SP‐induced currents reversed around −68 mV and displayed marked inward rectification. These data indicate that SP increased a ClC‐2‐type chloride conductance in MSNs, acting through NK1 receptors. SP also strongly increased glutamatergic responses in 49/89 MSNs. Facilitation of glutamatergic responses (which was observed both in MSNs directly affected by SP and in non‐affected ones) was reduced by application of L‐732,138, and fully blocked by coapplication of L‐732,138 and SB222200 (an NK3 receptor antagonists), showing that both NK1 and NK3 receptors were involved. SP‐induced facilitation of glutamatergic responses was accompanied by a marked decrease in paired‐pulse ratio, indicating a presynaptic mechanism of action. These data provide an electrophysiological correlate for the anatomically known connections between SP‐positive MSN terminals and the dendrites and somata of other MSNs.

Agrp neuron activity is required for alcohol-induced overeating

Alcohol intake associates with overeating in humans. This overeating is a clinical concern, but its causes are puzzling, because alcohol (ethanol) is a calorie-dense nutrient, and calorie intake usually suppresses brain appetite signals. The biological factors necessary for ethanol-induced overeating remain unclear, and societal causes have been proposed. Here we show that core elements of the brains feeding circuits—the hypothalamic Agrp neurons that are normally activated by starvation and evoke intense hunger—display electrical and biochemical hyperactivity on exposure to dietary doses of ethanol in brain slices. Furthermore, by circuit-specific chemogenetic interference in vivo, we find that the Agrp cell activity is essential for ethanol-induced overeating in the absence of societal factors, in single-housed mice. These data reveal how a widely consumed nutrient can paradoxically sustain brain starvation signals, and identify a biological factor required for appetite evoked by alcohol.

Accumbal D2 cells orchestrate innate risk-avoidance according to orexin signals

Excitation of accumbal D2 cells governs vital actions, including avoidance of learned risks, but the origins of this excitation and roles of D2 cells in innate risk-avoidance are unclear. Hypothalamic neurons producing orexins (also called hypocretins) enhance innate risk-avoidance via poorly understood neurocircuits. We describe a direct orexin→D2 excitatory circuit and show that D2 cell activity is necessary for orexin-dependent innate risk-avoidance in mice, thus revealing an unsuspected hypothalamus–accumbens interplay in action selection.Most species exhibit instinctive risk-avoidance, e.g., lab mice avoid predator smells despite having never encountered predators. Here the authors show how innate risk-avoidance arises from accumbal dopamine receptor neurons tuned by orexin signals.

Dual Nitrergic/Cholinergic Control of Short-Term Plasticity of Corticostriatal Inputs to Striatal Projection Neurons.

The ability of nitric oxide and acetylcholine to modulate the short-term plasticity of corticostriatal inputs was investigated using current-clamp recordings in BAC mouse brain slices. Glutamatergic responses were evoked by stimulation of corpus callosum in D1 and D2 dopamine receptor-expressing medium spiny neurons (D1-MSNs and D2-MSN, respectively). Paired-pulse stimulation (50 ms intervals) evoked depressing or facilitating responses in subgroups of both D1-MSNs and D2 MSNs. In both neuronal types, glutamatergic responses of cells that displayed paired-pulse depression were not significantly affected by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP; 100 μM). Conversely, in D1-MSNs and D2-MSNs that displayed paired-pulse facilitation, SNAP did not affect the first evoked response, but significantly reduced the amplitude of the second evoked EPSP, converting paired-pulse facilitation into paired-pulse depression. SNAP also strongly excited cholinergic interneurons and increased their cortical glutamatergic responses acting through a presynaptic mechanism. The effects of SNAP on glutamatergic response of D1-MSNs and D2-MSN were mediated by acetylcholine. The broad-spectrum muscarinic receptor antagonist atropine (25 μM) did not affect paired-pulse ratios and did not prevent the effects of SNAP. Conversely, the broad-spectrum nicotinic receptor antagonist tubocurarine (10 μM) fully mimicked and occluded the effects of SNAP. We concluded that phasic acetylcholine release mediates feedforward facilitation in MSNs through activation of nicotinic receptors on glutamatergic terminals and that nitric oxide, while increasing cholinergic interneurons’ firing, functionally impairs their ability to modulate glutamatergic inputs of MSNs. These results show that nitrergic and cholinergic transmission control the short-term plasticity of glutamatergic inputs in the striatum and reveal a novel cellular mechanism underlying paired-pulse facilitation in this area.

Serotonin inhibits low-threshold spike interneurons in the striatum

Key points  •  The striatum is the largest nucleus of the basal ganglia, a brain structure crucially involved in motor control. Recent results show that nitric oxide plays an important role in striatal pathophysiology. •  The activity of the striatum is modulated by extrinsic neurotransmitters such as serotonin, produced by specialised neurons located in the brainstem. •  This modulation is exerted through control of striatal interneurons. However, nitric oxide‐producing interneurons (NOS interneurons) have been difficult to investigate due to their rarity. •  Using transgenic mice in which NOS interneurons express green fluorescent protein, we found that NOS interneurons are strongly inhibited by serotonin. •  This inhibition is mediated by a specific class of serotonin receptors (5‐HT2C) causing an increase in a specific potassium conductance (KCNQ). •  These results cast light on the role of serotonin in the striatum, revealing that it tightly controls the activity of the only neuronal type that releases nitric oxide.