Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.     

Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.     

Immunocytochemical localization of the AMPA receptor subunits in the mesencephalic trigeminal nucleus of the rat

The mesencephalic trigeminal nucleus is composed of large (35-50 microns) pseudo-unipolar neurons. Closely associated with them are small (< 20 microns) multipolar neurons. An unique peculiarity of the pseudo-unipolar perikarya is that they receive synaptic input from various sources, which sets them apart from the dorsal root and cranial nerves sensory ganglia neurons. Whereas glutamate is the best neurotransmitter candidate in pseudo-unipolar neurons, glutamatergic input into them has not yet been reported. AMPA glutamate receptors are implicated in fast excitatory glutamatergic synaptic transmission. They have been localized ultrastructurally at postsynaptic sites. This study demonstrates that the pseudo-unipolar neurons of the mesencephalic trigeminal nucleus express AMPA glutamate receptor subunits, which indicates that these neurons receive glutamatergic input. Serial sections from the rostral pons and midbrain of Sprague-Dawley rats were immunostained with antibodies against C-terminus of AMPA receptor subunits: GluR1, GluR2/3, and GluR4. The immunoreaction was visualized with avidin-biotin-peroxidase/DAB for light and electron microscopy. With GluR1 antibody only the smallest multipolar neurons were recognized as immunopositive within the mesencephalic trigeminal nucleus. GluR2/3 stained the pseudo-unipolar neurons intensely within the entire rostro-caudal extent of the nucleus. In addition the former antibody stained small multipolar neurons within the mesencephalic trigeminal nucleus, though with somewhat larger dimensions than those immunoreactive for GluR1. Whereas the overall staining with GluR4 antibody was scant, those pseudo-unipolar neurons that were stained, were strongly stained. Furthermore, a considerable number of microglial cells within and surrounding the mesencephalic trigeminal nucleus displayed very intense immunoreactivity for GluR4. These results are discussed in the light of the glutamate receptor subunit composition.     

Origin of Ischemia-Induced Glutamate Efflux in the CA1 Field of the Gerbil Hippocampus: An In Vivo Brain Microdialysis Study

Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5-min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5- or 20-min ischemia was induced. When 5-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15-fold) was observed in the histologically normal CA1 field. When 20-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20-min ischemia, but remarkable ischemia-induced increases in glutamate were observed during the last 10 min of 20-min ischemia in both areas. An excessive increase in glutamate (100-fold) was observed during 20-min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5-min ischemia was induced, a significant increase in glutamate was observed during the last half of 5-min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia-induced glutamate accumulation in the CA1 field of the hippocampus during 5-min ischemia but contributes to the ischemia-induced increase in glutamate level during ischemia with a longer duration and that ischemia-induced efflux of glutamate in the CA1 field during 5-min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.     

5—羟色胺抑制谷氨酸对海马神经元的毒性作用

为探讨5-羟色胺（5-HT）对过量谷氨酸（glutamate,Glu)神经毒性的影响。观察了5-HT存在时,过量Glu对海马细胞存活率、海马脑片CA1区群锋电位（population spike,PS)及神经细胞膜Ga^2 电流的影响。结果发现：5-HT可明显提高过量Glu作用下海马神经细胞的存活率,减缓Glu对海马脑片CA1区PS的降低作用;在细胞膜上,5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用。在细胞膜上5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用,其机制可能在于5-HT与细胞膜上特定的受体结合,抑制了Glu诱导的Ca^2 内流。     

Hexamethonium, nicotinic receptor blocker, changes the neuronal reactions on glutamate in the medial septal area in vitro

Despite the great interest in studying the medial septal area, the interactions of its neurochemical systems are not yet clearly understood. The aim of this study was to elucidate the role of nicotinic receptors in the interaction of glutamatergic and cholinergic systems of the medial septal area. The effect of L-glutamate (1 microM) on septal neurons was studied under the application of hexamethonium, nicotinic cholinoreceptor blocker by using the method of extracellular recording of neuronal activity in brain slices of ground squirrels. The response of septal neurons to glutamate depended on the type of their initial activity and on the presence of pacemaker properties. For the first time, the ability of septal neurons to respond to glutamate with an increase in burst frequency was shown. The influence of hexamethonium on the neuronal activity was similar to that of glutamate. After a preliminary application of hexamethonium, the reactions of neurons to glutamate changed. The excitatory reactions were masked, while the inhibitory reactions became stronger. It was found that nicotinic cholinergic receptors modulated the reactions of MS-DB cells to glutamate and the expression of the oscillatory properties of the septal neuronal network.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

Brain cell metabolism is intimately associated with intracellular oxidation–reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca²⁺ abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.     

Increased Na+/Ca2+ Exchanger Activity Promotes Resistance to Excitotoxicity in Cortical Neurons of the Ground Squirrel (a Hibernator)

Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus). At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA) perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca²⁺]_i) upon glutamate insult. Moreover, we found that Na⁺/Ca²⁺ exchanger (NCX) activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage.     

Glutamate Transmission in the Rostral Ventrolateral Medullary Sympathetic Premotor Pathway

1. The aim of these studies was to test the hypothesis that glutamate is the principal excitatory neurotransmitter in the sympathetic premotor pathway from the rostral ventrolateral medulla (RVLM) to the sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord.2. Iontophoretic and pressure ejection of glutamate receptor agonists and antagonists was made onto antidromically identified splanchnic and adrenal SPNs before and during electrical stimulation of the RVLM in urethane/chloralose-anesthetized, artificially ventilated rats.3. SPNs were excited by both NMDA and non-NMDA glutamate receptor agonists. Blockade of glutamate receptors in the IML interrupted the ability of electrical activation of sympathetic premotor neurons in the RVLM to excite SPNs. Within the IML, antergradely labeled terminals of RVLM neurons were found to contain glutamate immunoreactivity and to make asymmetric synapses on local dendrites.4. These data support a significant role for glutamate neurotransmission in mediating the tonic and phasic excitation of SPNs by the sympathetic premotor pathway from the RVLM. It seems likely that stimulation of the RVLM produces glutamate release from both C1 and non-PNMT-containing axon terminals in the IML.     

垂体腺苷酸环化酶激活肽减轻谷氨酸对海马神经元的损伤并抑制胞内钙升高

对原代培养７～９ｄ的海马神经元给予谷氨酸处理,２４ｈ后,神经元的存活率降低。预先给予垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）能显著减少谷氨酸引起的海马神经元死亡。谷氨酸呈剂量依赖性增加海马神经元细胞内钙离子含量,ＰＡＣＡＰ能抑制谷氨酸引起的海马神经元细胞内钙离子浓度的升高,特异性ＰＡＣＡＰ Ⅰ型受体拮抗剂ＰＡＣＡＰ ６－３８能完全阻断ＰＡＣＡＰ减轻谷氨酸所致海马神经元损伤及降低谷氨酸所致神经元细胞内钙     

Effects of glutamate-induced excitotoxicity on calretinin-expressing neuron populations in the area postrema of the rat

We mapped the distribution of calretinin-immunoreactive neuron populations in a circumventricular organ of the rat, the area postrema, and investigated their sensitivity to excitotoxic stimuli mediated by subcutaneously administered monosodium glutamate. We were specifically interested to ascertain whether the presence of calretinin can, per se, confer an in vivo intrinsic resistance for area postrema neurons to glutamate excitotoxicity. We found that dense populations of calretinin-positive neurons displayed a subregional compartmentation in coronal sections of the area postrema along its rostrocaudal axis. We demonstrated that calretinin-positive neurons differ in their sensitivities to monosodium glutamate depending on their position within the area postrema. Neurons in the caudal area postrema were the most sensitive ones, while those in the rostral area postrema were spared of degeneration. We conclude that calretinin-positive neurons in the area postrema are not uniformly protected against glutamate excitotoxicity. It is possible that differences in the local concentrations of monosodium glutamate due to regional heterogeneities in density and permeability of the capillary bed rather than neuronal expression of calretinin account for the observed effects.     

Activation of the neurokinin-1 receptor by substance P triggers the release of substance P from cultured adult rat dorsal root ganglion neurons

Hypoxia and ischemia occur in the spinal cord when blood vessels of the spinal cord are compressed under pathological conditions such as spinal stenosis, tumors, and traumatic spinal injury. Here by using spinal cord slice preparations and patch-clamp recordings we investigated the influence of an ischemia-simulating medium on dorsal horn neurons in deep lamina, a region that plays a significant role in sensory hypersensitivity and pathological pain. We found that the ischemia-simulating medium induced large inward currents in dorsal horn neurons recorded. The onset of the ischemia-induced inward currents was age-dependent, being onset earlier in older animals. Increases of sensory input by the stimulation of afferent fibers with electrical impulses or by capsaicin significantly speeded up the onset of the ischemia-induced inward currents. The ischemia-induced inward currents were abolished by the glutamate receptor antagonists CNQX (20 μM) and APV (50 μM). The ischemia-induced inward currents were also substantially inhibited by the glutamate transporter inhibitor TBOA (100 μM). Our results suggest that ischemia caused reversal operation of glutamate transporters, leading to the release of glutamate via glutamate transporters and the subsequent activation of glutamate receptors in the spinal dorsal horn neurons.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

Glutamate evokes firing through activation of kainate receptors in chick accessory lobe neurons

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological and behavioral evidence suggests that neurons are present in ALs and the AL acts as a sensory organ of equilibrium during walking. Neurons in the outer layer of the AL consistently show glutamate-like immunoreactivity and neurons in the central region of the AL show glutamate receptor-like immunoreactivity. However, it is unknown how glutamate acts on the functional activity of AL neurons. In this study, we examined the effects of glutamate on the electrical activities of AL neurons using the patch clamp technique. There are two types of neurons among isolated AL neurons: spontaneously firing and silent neurons. Among silent neurons, 42 % of neurons responded to glutamate and generated repetitive firing. Kainate and glutamate in combination with the NMDA receptor antagonist, MK-801, also induced firing and evoked an inward current. On the other hand, the application of AMPA, NMDA or glutamate in combination with the non-NMDA receptor antagonist, CNQX, did not. These results indicate that chick AL neurons express functional kainate receptors to respond to glutamate and suggest that the glutamatergic transmission plays a role in excitatory regulation of AL neurons of the chick.     

THE EFFECT OF INTRAHIPPOCAMPAL KAINIC ACID INJECTIONS AND SURGICAL LESIONS ON NEUROTRANSMITTERS IN HIPPOCAMPUS AND SEPTUM

Local injection of kainic acid (2 μg) was accompanied by destruction of intrinsic neurons in the dorsal part of hippocampus. The lesion was accompanied by a 75% reduction in glutamate decarboxylase activity, a 60% reduction in the high affinity uptake of l -glutamate, a 40-60% reduction in the endogeneous levels of aspartate, glutamate and GABA and no changes in the activities of choline acetyltransferase or aromatic amino acid decarboxylase in the dorsal hippocampus. Unilateral destruction of neurons in the dorsal hippocampus was followed by a 20-40% reduction in the high affinity uptake of glutamate in lateral, but not in medial septum, on both sides. There was no reduction in choline acetyltransferase, glutamate decarboxylase or aromatic amino acid decarboxylase activities in the lateral or medial part of the septum. Transection of fimbria and superior fornix was accompanied by a severe reduction in choline acetyltransferase and aromatic amino acid decarboxylase activity in hippocampus, in the high affinity uptake of glutamate and in the endogenous level of glutamate in the lateral septum. The results are consistent with the concept that in the hippocampus kainic acid destroys intrinsic neurons and not afferent fibres. It seems therefore that all GABAergic fibres in the hippocampus belong to intrinsic neurons whereas glutamergic and aspartergic neurons belong partly to local neurons. The connection from the hippocampus to the lateral septum probably uses glutamate as a transmitter.     

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.