Inhibitory Neuronal Circuits Research Articles

5-HT3A receptor subunit is expressed in a subpopulation of GABAergic and enkephalinergic neurons in the mouse dorsal spinal cord

5-Hydroxytryptamine (5-HT)(3) receptors have been proposed to modulate nociception and pain responses at the spinal level. To gain insight into the cellular mechanism of 5-HT(3) receptors, we examined their expression in GABAergic and enkephalinergic (ENKergic) neurons in the spinal dorsal horn (SDH) using single-cell reverse transcription-polymerase chain reaction (RT-PCR). The glutamic acid decarboxylase (GAD)(67)-green fluorescent protein (GFP) knock-in mouse was used in which all GABAergic neurons were fluorescent. The general tissue RT-PCR results showed that 5-HT(3A) receptor subunit mRNA was present in the mouse SDH, while 5-HT(3B) receptor subunit was absent. Single-cell RT-PCR results showed that 76.2% (16/21) and 33.3% (7/21) of the total 5-HT(3A)-expressing neurons were positive for GAD(67) and preproenkephalin (PPE, a precursor of ENK), respectively. 5-HT(3A) receptor subunit was detected in 28.1% (16/57) of GABAergic neurons and 22.6% (7/31) of ENKergic neurons. About 40.4% (23/57) of GABAergic neurons expressed PPE mRNA. Of the neurons that co-express GAD(67) mRNA and PPE mRNA, about 22% expressed 5-HT(3A) mRNA. These observations indicate that 5-HT(3A) receptor co-localizes with GABA and ENK in the SDH, suggesting that serotonin may activate GABAergic and ENKergic neurons via 5-HT(3A) receptor subunit and therefore affect the release of GABA and ENK. The different cellular localization of 5-HT(3A) receptor subunit suggest the complex participation of this receptor in the inhibitory neuronal circuits of the SDH neurons.

Homeostatic control of the excitation–inhibition balance in cortical layer 5 pyramidal neurons

Homeostatic regulation in the brain is thought to be achieved through a control of the synaptic strength by close interactions between excitation and inhibition in cortical circuits. We recorded in a layer 5 pyramidal neuron of rat cortex the composite response to an electrical stimulation of various layers (2-3, 4 or 6). Decomposition of the global conductance change in its excitatory and inhibitory components permits a direct measurement of excitation-inhibition (E-I) balance. Whatever the stimulated layer was, afferent inputs led to a conductance change consisting of 20% excitation and 80% inhibition. Changing synaptic strengths in cortical networks by using a high-frequency of stimulation (HFS) protocol or a low-frequency of stimulation (LFS) protocol (classically used to induce long-term potentiation or long-term depression at the synaptic level) were checked in order to disrupt this balance. Application of HFS protocols in layers 2-3, 4 or 6, or of LFS protocols in layer 4 induced, respectively, long-term paralleled increases or long-term paralleled decreases in E and I which did not change the E-I balance. LFS protocols in layers 2-3 or 6 decreased E but not I and disrupted the balance. It is proposed that regulatory mechanisms might be mainly sustained by recurrent connectivity between excitatory and inhibitory neuronal circuits and by modulation of shunting GABA(A) inhibition in the layer 5 pyramidal neuron.

Excitability of human motor and visual cortex before, during, and after hyperventilation

In humans, hyperventilation (HV) has various effects on systemic physiology and, in particular, on neuronal excitability and synaptic transmission. However, it is far from clear how the effects of HV are mediated at the cortical level. In this study we investigated the effects of HV-induced hypocapnia on primary motor (M1) and visual cortex (V1) excitability. We used 1) motor threshold (MT) and phosphene threshold (PT) and 2) stimulus-response (S-R) curves (i.e., recruitment curves) as measures of excitability. In the motor cortex, we additionally investigated 3) the intrinsic inhibitory and facilitatory neuronal circuits using a short-interval paired-pulse paradigm. Measurements were performed before, during, and after 10 min of HV (resulting in a minimum end-tidal Pco(2) of 15 Torr). HV significantly increased motor-evoked potential (MEP) amplitudes, particularly at lower transcranial magnetic stimulation (TMS) intensities. Paired-pulse stimulation indicated that HV decreases intracortical inhibition (ICI) without changing intracortical facilitation. The results suggestthat low Pco(2) levels modulate, in particular, the intrinsic neuronal circuits of ICI, which are largely mediated by neurons containing gamma-aminobutyric acid. Modulation of MT probably resulted from alterations of Na(+) channel conductances. A significant decrease of PT, together with higher intensity of phosphenes at low stimulus intensities, furthermore suggested that HV acts on the excitability of M1 and V1 in a comparable fashion. This finding implies that HV also affects other brain structures besides the corticospinal motor system. The further exploration of these physiological mechanisms may contribute to the understanding of the various HV-related clinical phenomenona.

Distinct cerebral lesions in sporadic and ‘D90A’ SOD1 ALS: studies with [11C]flumazenil PET

Five to ten percent of amyotrophic lateral sclerosis (ALS) cases are associated with mutations of the superoxide dismutase-1 (SOD1) gene, and the 'D90A' mutation is associated with a unique phenotype and markedly slower disease progression (mean survival time 14 years). Relative sparing of inhibitory cortical neuronal circuits might be one mechanism contributing to the slower progression in patients homozygous for the D90A mutation (homD90A). The GABA(A) receptor PET ligand [11C]flumazenil has demonstrated motor and extra-motor cortical changes in sporadic ALS. In this study, we used [11C]flumazenil PET to explore differences in the pattern of cortical involvement between sporadic and genetically homogeneous ALS groups. Twenty-four sporadic ALS (sALS) and 10 homD90A patients underwent [11C]flumazenil PET of the brain. In addition, two subjects homozygous for the D90A mutation, but without symptoms or signs ('pre-symptomatic', psD90A), also underwent imaging. Results for each group were compared with those for 24 healthy controls of similar age. Decreases in the binding of [11C]flumazenil in the sALS group were found within premotor regions, motor cortex and posterior motor association areas. In the homD90A group of ALS patients, however, decreases were concentrated in the left fronto-temporal junction and anterior cingulate gyrus. In the two psD90A subjects, a small focus of reduced [11C]flumazenil binding at the left fronto-temporal junction was seen, similar to the pattern seen in the clinically affected patients. Within the sALS group, there was no statistically significant association between decreases in cortical [11C]flumazenil binding and revised ALS functional rating scale (ALSFRS-R score), whereas the upper motor neuron (UMN) score correlated with widespread and marked cortical decreases over the dominant hemisphere. In the homD90A group, there was a stronger statistical association between reduced cortical [11C]flumazenil binding and the ALSFRS-R, rather than the UMN, score, and also with disease duration. This study provides evidence for differences in the distribution of reduced cortical [11C]flumazenil binding in homD90A compared with sALS patients. We hypothesize that this might reflect differences in cortical neuronal vulnerability.

Inhibitory synapses in the developing auditory system are glutamatergic

Activity-dependent synapse refinement is crucial for the formation of precise excitatory and inhibitory neuronal circuits. Whereas the mechanisms that guide refinement of excitatory circuits are becoming increasingly clear, the mechanisms guiding inhibitory circuits have remained obscure. In the lateral superior olive (LSO), a nucleus in the mammalian sound localization system that receives inhibitory input from the medial nucleus of the trapezoid body (MNTB), specific elimination and strengthening of synapses that are both GABAergic and glycinergic (GABA/glycinergic synapses) is essential for the formation of a precise tonotopic map. We provide evidence that immature GABA/glycinergic synapses in the rat LSO also release the excitatory neurotransmitter glutamate, which activates postsynaptic NMDA receptors (NMDARs). Immunohistochemical studies demonstrate synaptic colocalization of the vesicular glutamate transporter 3 with the vesicular GABA transporter, indicating that GABA, glycine and glutamate are released from single MNTB terminals. Glutamatergic transmission at MNTB-LSO synapses is most prominent during the period of synapse elimination. Synapse-specific activation of NMDARs by glutamate release at GABAergic and glycinergic synapses could be important in activity-dependent refinement of inhibitory circuits.

Contact-associated neurite outgrowth and branching of immature cortical interneurons.

When juvenile interneurons arrive at the cortical environment following tangential migration, they are faced with the task of positioning themselves in cortical space in preparation for local circuit wiring. This includes integration into different cortical layers and cessation of migration at various positions to ensure adequate coverage. Little is known about the signals or mechanisms that initiate a conversion from the migratory phenotype to the arborization phenotype. This study looks at the immediate changes in interneuron morphology after culturing for 24 h in a three-dimensional collagen gel. Immature interneurons taken from different stages of corticogenesis showed increased neurite branching and outgrowth after interneuronal contacts were made. These responses were suppressed in the presence of Slit and brain-derived neurotrophic factor (BDNF) if the interneurons were sourced from early to mid-stages of corticogenesis. However, interneurons taken from the late period of corticogenesis responded to Slit and BDNF by increasing branching and neurite outgrowth. These results suggest an initial interneuronal cell contact as a stimulus for propagating neuronal arborization that may lead to the formation of inhibitory neuronal circuits. In addition, we have identified the late corticogenetic period when interneurons are most sensitive to the neurite promoting effects of Slit and BDNF.

Changes in intracortical excitability induced by stimulation of wrist afferents in man.

1. Inhibitory and facilitatory neuronal circuits may be explored in the human motor cortex by double pulse transcranial magnetic stimulation (TMS). At short interstimulus intervals (2-5 ms), conditioned motor-evoked potentials (MEPs) are reduced (intracortical inhibition, ICI), whereas they are facilitated at longer interstimulus intervals (8-25 ms; intracortical facilitation, ICF). The aim of this study was to investigate the effects of homonymous and antagonist nerve stimulation on the intracortical inhibition and facilitation in the cortical areas that control the wrist extensor and flexor radialis muscles. 2. Sixteen subjects were asked to contract either their wrist extensor or flexor muscles. The MEP evoked by a test TMS (at 1.2 x MEP threshold) and recorded in the target muscle was then conditioned by subthreshold TMS (at 0.8 x MEP threshold) 2 and 14 ms before the test TMS. The median and radial nerves were stimulated at 0.8 x motor threshold (MT). 3. In both flexor and extensor muscles, antagonist nerve stimulation 40 ms before the test TMS decreased ICI and increased ICF. In contrast, homonymous nerve stimulation had no effect on ICI and ICF. 4. The intensity of the antagonist nerve stimulation required to alter ICI and ICF was as low as 0.6 x MT, which suggests that thick diameter afferents may be involved. The nerve stimulation had to be applied 35-45 ms prior to the test TMS to alter significantly the intracortical excitability. 5. Cutaneous afferents were probably not responsible for the alterations of intracortical excitability, since cutaneous stimulation had no effect on either ICI or ICF at the investigated intervals. 6. The present data suggest that antagonist muscular afferent inputs may evoke reciprocal facilitation or disinhibition at the cortical level. This pattern of antagonist sensory afferent effects may be of significance for control of the wrist extensor and flexor muscles when used as synergists during manipulatory finger movements and gripping tasks.

Pharmacological evidence of inhibitory and disinhibitory neuronal circuits in dorsal cochlear nucleus.

The dorsal cochlear nucleus (DCN) is rich in both glycine and GABA inhibitory neurotransmitter systems, and the response properties of its principal cells (pyramidal and giant cells) are strongly shaped by inhibitory inputs. For example, DCN principal cells often display highly nonmonotonic (so-called type IV) input-output functions in response to best-frequency (BF) tones. In this study, the inhibitory inputs onto the principal cell types and onto response types of known inhibitory interneurons were compared before and during iontophoretic application of the glycine- and GABA(A)-receptor antagonists, strychnine and bicuculline. Strychnine eliminates the central (on-BF) inhibitory area in type IV units, resulting in monotonic BF rate-level curves. Unexpectedly, bicuculline primarily enhances inhibition in principal-cell types; for example, type IV units are inhibited at lower sound levels in the presence of bicuculline. Principal cell types with weaker inhibitory inputs (type IV-T and type III units) are more strongly inhibited in the presence of bicuculline and usually are converted into type IV units. This enhancement of on-BF inhibition by bicuculline suggests a disinhibitory process involving GABA(A) action on a non-GABA(A)ergic inhibitory pathway. This latter pathway is probably glycinergic and involves type II units (deep-layer vertical cells) and/or complex-spiking units (superficial cartwheel cells) because both of these unit types are disinhibited by bicuculline. One intrinsic GABA(A) source could be the superficial stellate cells in DCN because bicuculline partly blocks the inhibition evoked by somatosensory-stimulated activation of the superficial granule-cell circuitry in DCN. Taken together, the results suggest that glycinergic circuits mediate directly the inhibition of DCN principal cells, but that GABA(A)ergic circuits modulate the strength of the inhibition.

Reversal of GABA Actions by Neuronal Trauma

GABA is the most prevalent inhibitory transmitter in the adult brain where it reduces neuronal activity mainly by opening chloride channels and hyperpolarizing the membrane potential. Surprisingly, after some types of neuronal trauma, GABA exerts a different action, depolarizing the membrane potential, raising cytoplasmic calcium levels, and increasing neuronal activity. After trauma, GABA can generate cytoplasmic calcium rises even larger than those elicited by the excitatory transmitter glutamate. Large GABA-mediated increases in intracellular calcium could be toxic. Furthermore, if inhibitory neuronal circuits switched to excitatory actions, maladaptive signaling may be generated in affected pathways. These depolarizing actions of GABA after injury are similar to GABA's function in early neuronal development. Neuronal injury, thus, may generate a recapitulation of GABA's role in ontogeny. NEUROSCIENTIST 3:281–286, 1997

Inhibitory neurons in the human epileptogenic temporal neocortex. An immunocytochemical study.

Immunocytochemical methods were used to study alterations in inhibitory neuronal circuits in human neocortex resected during surgical treatment of intractable temporal epilepsy associated or not with brain tumours. The epileptogenic cortex was characterized and divided into spiking or non-spiking zones by intraoperative electrocorticography (ECOG). The resected cortex was cut into blocks, sectioned and stained immunocytochemically for visualization of glutamic acid decarboxylase (GAD), the calcium-binding protein, parvalbumin (PV) and glial fibrillary acidic protein (GFAP). A variety of alterations in cortical neuronal circuits as revealed by immunocytochemical and histological methods were found. Similar alterations in inhibitory neuronal circuits appear to occur independently of the primary epileptogenic site and pathology associated with epilepsy, which suggests that there is possibly a common basic underlying mechanism that leads to seizure activity. These changes were apparently unrelated to ECOG findings at surgery, which bring into question the value of the use of interictal epileptic discharges recorded by ECOG to guide cortical resections. The most conspicuous and common change was the loss of chandelier cells. The finding that these cells are among the most vulnerable types of GABAergic interneurons in the epileptogenic temporal cortex indicates that they might be of great functional importance, since the axon terminals of chandelier cells are likely to exert powerful regulation of impulse generation in cortical pyramidal cells. Therefore, these cells might represent a key component in the aetiology of human epilepsy.

The action of peptides on the mudpuppy electroretinogram (ERG)

Two neuropeptide substances were applied to the mudpuppy retina while the electroretinogram (ERG) was recorded. A low concentration of somatostatin (10 −9 m) was found to be a potent agent in increasing the amplitudes of all the oscillatory potentials (OPs) of the ERG. There was no appreciable change of the threshold sensitivity or the stimulus response curve of the b-wave. The highest concentration tested (10 −6 m) reduced the first OP to about the same amplitude as at the start of the experiment and attenuated the later OPs. A decrease of the suprathreshold b-wave was induced by the highest concentrations (10 −7, 10 −6 m) of somatostatin. A low concentration of substance P (10 −11 m) selectively and differentially decreased the earlier OPs (O 1O 2). Higher concentrations (10 −10, 10 −9 m) diminished the earlier OPs further, reduced the later OPs and decreased the supra-threshold a- and b-waves. The results support previous suggestions that the OPs reflect activities in feedback circuits initiated by the amacrines and indicate that somatostatin and substance P through separate mechanisms seem to interact with the inhibitory neuronal circuits which have been suggested to give rise to the OPs. Secondly, in agreement with previous work, the OPs appear to have a different origin from the b-wave. Thirdly, two separate classes of amacrines, each with a different transmitter, seem to be associated with different physiological roles.

Some Physiological Aspects of the Basal Ganglia

The caudate efferent fibers to the globus pallidus are inhibitory in nature. The pallidothalamic fibers are also inhibitory, exerting monosynaptic IPSPs on the thalamic cells located anteroventrally to the ventrolateral nucleus. Therefore, these two inhibitory neuronal circuits make a double inhibitory chain. To operate the inhibitory circuit, excitatory input to the pallidum is necessary. Stimulation of the ipsilateral brain stem including lemniscus medialis, mesencephalic reticular formation and some other structures, produces short latency excitation of the pallidal cells.