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Enrichment of mGluR7a in the presynaptic active zones of GABAergic and non-GABAergic terminals on interneurons in the rat somatosensory cortex.

The release of glutamate and GABA is modulated by presynaptic metabotropic glutamate receptors (mGluRs). We used immunocytochemical methods to define the location of the group III receptor mGluR7a in glutamatergic and GABAergic terminals innervating GABAergic interneurons and pyramidal cells. Immunoreactivity for mGluR7a was localized in the presynaptic active zone of both identified GABAergic and presumed glutamatergic terminals. Terminals innervating dendritic spines showed a variable level of receptor immunoreactivity, ranging from immunonegative to strongly immunopositive. The frequency of strongly mGluR7a positive terminals innervating the soma and dendrites of mGluR1 alpha/somatostatin-expressing interneurons was very high relative to other neurons. On dendrites that received mGluR7a-enriched glutamatergic innervation, at least 80% of GABAergic terminals were immunopositive for mGluR7a. On such dendrites virtually all (95%) vasoactive intestinal polypeptide (VIP) positive (GABAergic) terminals were enriched in mGluR7a. The targets of VIP/mGluR7a-expressing terminals were mainly (88%) mGluR1 alpha-expressing interneurons, which were mostly somatostatin immunopositive. Parvalbumin positive terminals were immunonegative for mGluR7a. Some parvalbumin immunoreactive dendrites received strongly mGluR7a positive terminals. The subcellular location, as well as the cell type and synapse-specific distribution of mGluR7a in isocortical neuronal circuits, is homologous to its distribution in the hippocampus. The specific location of mGluR7a in the presynaptic active zone of both glutamatergic and GABAergic synapses may be related to the proximity of calcium channels and the vesicle fusion machinery. The enrichment of mGluR7a in the main GABAergic, as well as in the glutamatergic, innervation of mGluR1 alpha/somatostatin-expressing interneurons suggests that their activation is under unique regulation by extracellular glutamate.

Animals↗

Fast-spike interneurons and feedforward inhibition in awake sensory neocortex.

'Fast-spike' interneurons of layer 4 mediate thalamocortical feedforward inhibition and can, with some confidence, be identified using extracellular methods. In somatosensory barrel cortex of awake rabbits, these 'suspected inhibitory interneurons' (SINs) have distinct receptive field properties: they respond to vibrissa displacement with very high sensitivity and temporal fidelity. However, they lack the directional specificity that is clearly seen in most of their ventrobasal thalamocortical afferents. Several lines of evidence show that layer-4 SINs receive a potent and highly convergent and divergent functional input from topographically aligned thalamocortical neurons. Whereas the unselective pooling of convergent thalamocortical inputs onto SINs generates sensitive and broadly tuned inhibitory receptive fields, the potent divergence of single thalamocortical neurons onto many SINs generates sharply synchronous (+/-1 ms) activity (because of coincident EPSPs). Synchronous discharge of these interneurons following thalamocortical impulses will generate a synchronous feedforward release of GABA within the barrel. Thalamocortical impulses will, therefore, generate only a brief 'window of excitability' during which spikes can occur in the post-synaptic targets of fast-spike interneurons. This fast, synchronous, highly sensitive and broadly tuned feed-forward inhibitory network is well suited to suppress spike generation in spiny neurons following all but the most optimal feedforward excitatory inputs.

Action Potentials↗

Ringo, Doty, Demeter and Simard, Cerebral Cortex 1994;4:331-343: a proof of the need for the spatial clustering of interneuronal connections to enhance cortical computation.

It has been argued that an important principle driving the organization of the cerebral cortex towards local processing has been the need to decrease time lost to interneuronal conduction delay. In this paper, I show for a simplified model of the cerebral cortex, using analytical means, that if interneuronal conduction time increases proportional to interneuronal distance, then the only way to increase the numbers of synaptic events occurring in a fixed finite time period is to spatially cluster interneuronal connections.

Cerebral Cortex↗

Preferential origin and layer destination of GAD65-GFP cortical interneurons.

To identify the origin and track the migratory pathway of specific subpopulations of GABAergic interneurons, we studied tangential migration in a recently developed GAD65-GFP transgenic mouse strain. First, we used immunohistochemical methods to characterize the expression of specific neurochemical markers in the GAD65-GFP neurons. Then, organotypic cultures were used in combination with birth-dating studies to determine the time of generation, place of origin and migratory route of these cells. From E14 to E15, the highest density of GAD65-GFP cells was seen in the lower intermediate zone; however, at later stages more GAD65-GFP cells were observed in the subventricular zone. Migratory GAD65-GFP cells express GAD65, but not calretinin or reelin. Surprisingly, only 4% were calbindin immunopositive. At P21, GAD65-GFP cells were found predominantly in layers II-III and expressed calretinin and neuropeptide Y. Remarkably, almost all cholecystokinin-positive but very few parvalbumin-positive neurons expressed GFP. In vitro studies demonstrated that the caudal ganglionic eminence gives rise to a large proportion of GAD65-GFP interneurons and in vivo birth-dating experiments showed that GAD65-GFP interneurons in supragranular layers are born at late embryonic development. Taken together these results support the idea that the destination layer of GABAergic interneurons is closely linked to their place of origin and time of generation.

Animals↗

Cell-type specific alterations of cortical interneurons in schizophrenic patients.

In the human brain, cortical GABAergic interneurons represent an important population of local circuit neurons responsible for the intrinsic modulation of neuronal information and have been supposed to be involved in the pathophysiology of schizophrenia. We conducted a quantitative study on the differentiated three-dimensional morphological structure of two types of parvalbumin-immunoreactive interneurons in the anterior cingulate cortex (ACC) of schizophrenic patients versus controls. While type A interneurons ('small bipolar cells') showed a significant reduction of their soma size in schizophrenics, type B interneurons ('small multipolar cells') of schizophrenic patients exhibited a marked decrease in the extent of their dendritic system. These results further support the assumption of a considerable significance of the ACC, an important limbic relay centre, for the etiopathogenesis of schizophrenic psychoses.

Cerebral Cortex↗

Ablation of striatal interneurons influences activities of entopeduncular neurons.

To characterize modulatory effects of striatal interneurons upon the output nucleus of the basal ganglia, we ablated striatal interneurons that express substance P receptors by using local injection of a selective neurotoxin, substance P-saporin, in rats. We then made extracellular recordings of the activity of entopeduncular neurons and examined their responses to stimulation in the motor cortex. In the interneuron-ablated animals, the spontaneous discharge rate of entopeduncular neurons was significantly decreased, and the proportion of entopeduncular neurons showing responses to cortical stimulation was significantly larger, in comparison with intact animals. It is suggested that striatal interneurons expressing substance P receptors are important for motor control mechanisms mediated by the cortico-basal ganglia pathways.

Action Potentials↗

Irreversible loss of a subpopulation of cortical interneurons in the absence of glutamatergic network activity.

In the cerebral cortex of mammals, gamma-aminobutyric acid (GABA)ergic neurons represent 15-25% of all neurons, depending on the species and area being examined. Because converging evidence suggests that activity may play an important role in the neuritic maturation and synaptic function of GABAergic neurons, it is feasible that activity plays a role in the regulation of the proportion of GABAergic neurons. Here we provide direct evidence that early in cortical development activity blockade may deplete the network of a subpopulation of GABA immunoreactive neurons characterized by their small size and late generation in vitro. In a period of time coinciding with the emergence of synchronous network activity, the survival and morphological differentiation of GABAergic neurons was influenced by long-term blockade of synaptic activity. While GABA(A) receptor antagonists had a minor promoting effect on interneuronal survival during the second week in vitro, antagonists of ionotropic glutamate receptors strongly impaired survival and differentiation of immature GABAergic interneurons. Interneuronal loss was more severe when N-methyl-D-aspartate receptors were blocked than after blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptors. The decrease in the density of GABAergic neurons was irreversible, but could be prevented by the simultaneous addition of brain-derived neurotrophic factor (BDNF). These results suggest that there is a narrow time window during neocortical development when glutamatergic activity, and specially NMDA receptor stimulation, is crucial to assure survival and maturation of a subpopulation of late developing GABAergic interneurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerability.

Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+-free medium, NMDA (10 microM; 3-5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 microM) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mM) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition.

Anesthetics, Local↗

Functional nicotinic ACh receptors on interneurones in the rat hippocampus.

1. Neuronal nicotinic ACh receptors (nAChRs) were studied in the rat hippocampal slice preparation using whole-cell patch-clamp recording techniques. 2. Responses to ACh (100 microM) were detected on inhibitory interneurones in the Ca1 field of the hippocampus proper and in the dentate gyrus, but not on principal excitatory neurones in either region. The different neuronal types were identified based on their morphology and location. 3. ACh excited interneurones in the hippocampus and dentate gyrus in current-clamp recordings. In voltage-clamp recordings, ACh-activated inward currents were recorded from interneurones in the presence of blockers of synaptic transmission and the muscarinic ACh receptor antagonist atropine. The zero current potential for this response to ACh was near 0 mV. 4. The effect of ACh was mimicked by the nAChR-selective agonists nicotine (100 microM) and 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 microM). The response to ACh was reversibly antagonized by the neuronal nAChR antagonist mecamylamine (10 microM). The nAChR alpha 7 subunit-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM) also inhibited the response to ACh. 5. These observations demonstrate the presence of functional nAChRs on inhibitory interneurones in the rat hippocampus. Thus, a novel mechanism by which ACh can regulate neuronal activity in the hippocampus is revealed.

Animals↗

Spinal interneuronal systems: identification, multifunctional character and reconfigurations in mammals.

This review focuses on the flexibility of operation of spinal interneuronal networks and their multifunctional character in mammals. It concerns, in particular, two ways in which spinal interneuronal networks may be functionally reorganised, namely by modulating the synaptic actions of primary afferents by monoamines and by GABAergic presynaptic inhibition. The evidence will be reviewed for topographical and target-related differences in modulatory effects in various interneuronal networks and these will be related to differences in the intrinsic properties of different functional types of interneurones in these networks and to the role played by them.

Animals↗

Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons?

PURPOSE: Neuronal migration disorders (NMD) are often associated with therapy-resistant epilepsy. In human cerebral cortex, this hyperexcitability has been correlated with a loss of inhibitory interneurons. We used a rat model of focal cortical NMD (microgyria) to determine whether the expression of epileptiform activity in this model coincides with a decrease in inhibitory interneurons. METHODS: In 2-to 4-month-old rats, the density of interneurons immunoreactive for gamma-aminobutyric acid (GABA), calbindin, and parvalbumin was determined in fronto-parietal cortex in nine 200-microm-wide sectors located up to 2.5 mm lateral and 2.0 mm medial from the lesion center in primary parietal cortex (Par1). Quantitative measurements in homotopic areas of age-matched sham-operated rats served as controls. RESULTS: The freeze lesion performed in newborn rat cortex resulted in adult rats with a microgyrus extending in a rostro-caudal direction from frontal to occipital cortex. The density of GABA-and parvalbumin-positive neurons in fronto-parietal cortex was not significantly different between lesioned and control animals. Only the density of calbindin-immunoreactive neurons located 1.0 mm lateral and 0.5 mm medial from the lesion was significantly (Student t test, p < 0.05) larger in freeze-lesioned rats (5,817 +/- 562 and 6,400 +/- 795 cells per mm3, respectively; n = 12) compared with measurements in homotopic regions in Par1 cortex of controls (4,507 +/- 281 and 4, 061 +/- 319 cells per mm3, respectively; n = 5). CONCLUSIONS: The previously reported widespread functional changes in this model of cortical NMD are not related to a general loss of inhibitory interneurons. Other factors, such as a decrease in GABA receptor density, modifications in GABAA receptor subunit composition, or alterations in the excitatory network, e.g., an increase in the density of calbindin-immunoreactive pyramidal cells, more likely contribute to the global disinhibition and widespread expression of pathophysiological activity in this model of cortical NMD.

Adult↗

Neuroadaptations of Cdk5 in cholinergic interneurons of the nucleus accumbens and prefrontal cortex of inbred alcohol-preferring rats following voluntary alcohol drinking.

BACKGROUND: Neurobiological studies have identified brain areas and related molecular mechanisms involved in alcohol abuse and dependence. Specific cell types in these brain areas and their role in alcohol-related behaviors, however, have not yet been identified. This study examined the involvement of cholinergic cells in inbred alcohol-preferring rats following 1 month of alcohol drinking. Cyclin-dependent kinase 5 (Cdk5) immunoreactivity (IR), a marker of neuronal plasticity, was examined in cholinergic neurons of the nucleus accumbens (NuAcc) and prefrontal cortex (PFC) and other brain areas implicated in alcohol drinking, using dual immunocytochemical (ICC) procedures. Single Cdk5 IR was also examined in several brain areas implicated in alcohol drinking. METHODS: The experimental group self-administered alcohol using a 2-bottle-choice test paradigm with unlimited access to 10% (v/v) alcohol and water for 23 h/d for 1 month. An average of 6 g/kg alcohol was consumed daily. Control animals received identical treatment, except that both bottles contained water. Rats were perfused and brain sections were processed for ICC procedures. RESULTS: Alcohol drinking resulted in a 51% increase in Cdk5 IR cholinergic interneurons in the shell NuAcc, while in the PFC there was a 51% decrease in the percent of Cdk5 IR cholinergic interneurons in the infralimbic region and a 46% decrease in Cdk5 IR cholinergic interneurons in the prelimbic region. Additionally, single Cdk5 IR revealed a 42% increase in the central nucleus of the amygdala (CNA). CONCLUSIONS: This study identified Cdk5 neuroadaptation in cholinergic interneurons of the NuAcc and PFC and in other neurons of the CNA following 1 month of alcohol drinking. These findings contribute to our understanding of the cellular and molecular basis of alcohol drinking and toward the development of improved region and cell-specific pharmacotherapeutic and behavioral treatment programs for alcohol abuse and alcoholism.

Adaptation, Physiological↗

Estimation of the projection frequencies of single inhibitory interneurones to motoneurones in the cat spinal cord.

An estimate has been deduced for the percentage of motoneurones in the lumbosacral spinal cord inhibited by single interneurones mediating group I non-reciprocal inhibition. The estimate is derived from data for the percentage of interneurones discharged, and percentage of motoneurones inhibited, by the same group I afferents. These experimental data are compared with theoretical curves for the percentage of motoneurones in which inhibition would be expected following discharges in various numbers of interneurones, for different values of projection frequency. It is estimated that each interneurone projects to less than 5% of target motoneurones.

Animals↗

Quantitative aspects of responses in trigeminal relay neurones and interneurones following mechanical stimulation of sinus hairs and skin in the cat.

1. Stimulus-response relationships in discharges of trigeminal relay- and interneurones were investigated in the barbiturate anaesthetized cat using controlled sinus hair or skin displacements.2. In comparison with discharges in slowly adapting primary afferent fibres the responses in all higher order neurones were considerably reduced in firing rate and often revealed modifications suggesting the interaction of mechanisms actively modulating the afferent input.3. In relay neurones with or without a tonic discharge component the ;dynamic on' response during a trapezoidal displacement of sinus hairs was found to be determined entirely or predominantly by the movement velocity and to be independent of the deflexion angle of a stimulus. In contrast, the static response in tonic relay neurones was determined by both the movement velocity and the displacement amplitude.4. Spatial summation of afferent input caused either only quantitative changes in the responses of relay neurones leaving the general discharge properties unaltered or caused both qualitative and quantitative changes in the responses.5. Interneurones consisted of two functional groups. In about 25% of them the responses were not or only slightly dependent on the intensity of the applied stimulus, often burstlike and of an all or nothing character. In the second group of interneurones the responses showed a quantitative dependence on the applied stimuli. In this group of interneurones responses often increased with the spatial extension of the peripheral stimulus revealing spatial summation of the afferent input.

Animals↗

Conductance increases produced by glycine and gamma-aminobutyric acid in lamprey interneurones.

1. Conductances of individual neurones in the isolated lamprey spinal cord were measured with separate intracellular electrodes for recording potentials and for passing current pulses during application of glycine or GABA (0.1-1.0 MM) in Ca-free bathing fluid. Large, reversible increases in conductance were produced in giant interneurones by both amino acids, but Müller axons and sensory dorsal cells were unaffected. 2. Conductance increases produced by glycine and by GABA were selective for Cl. Both conductance increases were linearly related to external Cl concentrations and repeated exposure to the amino acids in Cl-free fluid progressively reduced the conductance increases to less than 1% of their values in normal Cl. 3. Strychnine was a competitive antagonist of glycine, while GABA was antagonized competitively by bicuculline and non-competitively by picrotoxin. 4. The sensitivity of giant interneurones to glycine and GABA increased at low temperatures, in Na-free fluid, and after repeated exposure to the amino acids. Sensitization may have been produced by inhibition of uptake mechanisms for glycine and GABA in the spinal cord. 5. Discharges of interneurones recorded extracellularly were inhibited by bath-applied glycine and GABA, but directly elicited action potentials of axons were unaffected. Strychnine and Cl-free fluid in the presence of Ca produced seizures in lamprey spinal cord. 6. The conclusions of these experiments are that different receptors for glycine and for GABA are present on giant interneurones, that glycine is the better candidate for an inhibitory transmitter in the lamprey spinal cord, and that GABA produces effects similar to those which have been well studied in arthropod muscle.

Action Potentials↗

Common interneurones in reflex pathways from group 1a and 1b afferents of knee flexors and extensors in the cat.

1. Input from group I afferents of knee flexors and extensors to interneurones in Rexed's laminae V-VI in the cat spinal cord was analysed by use of intracellular recording and electrical stimulation of the nerves to differentiate between group Ia and Ib synaptic actions. The aim was to find out if these interneurones may mediate synaptic actions of both group Ia and Ib afferents. 2. 28% of the forty-nine neurones analysed were excited by both group Ia and group Ib afferents; 32% were inhibited by both and 35% were excited by the one and inhibited by the other. Taking into account all of these actions, input from both subgroups of group I afferents was found in nearly 60% of neurones. Most were also excited and/or inhibited by group I afferents of ankle and toe extensors. 3. Selective (excitatory and/or inhibitory) input from Ia afferents was found in 18% and from Ib afferents in 22% of the neurones. 4. Excitation was evoked from Ia afferents of either knee flexors or extensors but not from both. In several of the neurones Ia i.p.s.p.s were, however, evoked from both posterior biceps-semitendinosus and quadriceps. 5. Intracellular staining with horseradish peroxidase revealed axonal projections of laminae V-VI interneurones to motor nuclei as well as to the intermediate zone, ipsilateral as well as contralateral. No correlation was found between patterns of input from group I afferents and axonal projections, and interneurones co-excited by Ia and Ib afferents were among these with different axonal projections.

Afferent Pathways↗

Convergence of Ia fibres from synergistic and antagonistic muscles onto interneurones inhibitory to soleus in humans.

1. The possibility that Ia afferent fibres from the gastrocnemius medialis (GM) and from the tibialis anterior (TA) muscle could converge on to a single interneuronal pool inhibitory to the soleus motoneurones was investigated. 2. The soleus H reflex, evoked by tibial nerve stimulation in the popliteal fossa, was conditioned by separate or combined stimulation of the nerves to the GM or TA muscles. Stimulus intensity was below the motor threshold (MTh), and the conditioning-test intervals were such as to evoke short-latency inhibition of the soleus H reflex. Care was taken to avoid current spread and artifacts connected with the closeness in time and space of the conditioning and test stimuli. 3. Separate stimulation of both GM and TA nerves was able to induce significant inhibitory effects on the H reflex amplitude at stimulus strengths larger than 0.75 x MTh, on the average. Combined stimulation of the two nerves was able to reduce the H reflex at lower stimulus strengths, at which either nerve was ineffective alone. 4. Conditioning stimulus strengths close to the MTh reduced the H reflex to approximately 80% of the control value, both on single and combined stimulation, i.e. saturation of the inhibitory effect was found. 5. By extrapolating the regression line through the normalized data from all subjects, it was assumed that the smallest stimulus strength necessary to drive the inhibitory interneurones to threshold was, on the average, 0.5 and 0.6 x MTh, on combined and separate nerve stimulation, respectively. 6. Tonic voluntary activation of the soleus abolished the inhibitory effects of both separate and combined stimulations. This was tested on the H reflex, on the rectified and averaged EMG, and on the peristimulus histogram of single motor unit discharge. 7. The findings strongly suggest the existence of spatial summation of the effects from GM and TA muscle at the level of a single interneuronal pool. Most probably, the responsible afferent fibres are group I spindle afferents, and the interneurones those mediating the reciprocal inhibition. The data do not support the notion of parallel pathways, exclusive to each nerve.

Adult↗

Cholinergic excitation of GABAergic interneurons in the rat hippocampal slice.

1. Intracellular recordings were made from CA1 pyramidal cells in the rat hippocampal slice to study the cholinergic modulation of GABAergic inhibition. The cholinergic receptor agonist, carbamylcholine (carbachol), depressed evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs), but enhanced small spontaneously occurring membrane potential fluctuations that resembled IPSPs. Both atropine (1 microM) and picrotoxin (25-60 microM) abolished the small fluctuations. 2. Recording from cells with potassium or caesium chloride (KCl or CsCl)-filled microelectrodes enhanced and inverted spontaneous Cl(-)-dependent GABAA-mediated IPSPs. These events appeared to result from the spontaneous firing of GABAergic interneurons since they could be inhibited by picrotoxin or bicuculline and nearly eliminated by tetrodotoxin. 3. Muscarinic acetylcholine (ACh) receptor activation significantly increased the frequency of spontaneous-activity-dependent IPSPs from 1.7 +/- 0.4 s (mean +/- S.E.M.) in control saline to 7.0 +/- 1.1 s in carbachol (10-50 microM)-containing saline, although evoked IPSPs were inhibited. All effects of carbachol were completely reversed by atropine. 4. The increase in frequency of spontaneous IPSPs observed in carbachol was not secondary to changes in the postsynaptic cell and was not blocked by high doses of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5-10 microM) and 2-amino-5-phosphonovaleric acid (APV, 10-20 microM), which abolished evoked excitatory transmission. Amplitude histograms showed an increase in mean size as well as of frequency of spontaneous IPSCs in carbachol. 5. Stimulation of cholinergic afferents in stratum oriens in the presence of the acetylcholinesterase inhibitor eserine (1 microM) also increased spontaneous IPSP frequency, and the time course of this response was similar to that of the muscarinic slow EPSP. Postsynaptic factors or the activation of glutamatergic excitatory pathways could not account for this effect. 6. Evoked monosynaptic IPSCs in CNQX and APV were diminished by carbachol. 7. We conclude that GABAergic inhibitory interneurons possess muscarinic receptors, that activation of these receptors increases the excitability of the interneurons and that synaptically released ACh increases interneuronal activity. Cholinergic reduction of the monosynaptic IPSC may point to additional complexity in cholinergic regulation of the GABA system.

2-Amino-5-phosphonovalerate↗