The Long-Term Effects of Early Postnatal Stress on Cognitive Abilities and Expression of Genes of the Glutamatergic System in Mice

Stressing events in the early period of life affect neuronal plasticity and cognitive functions in adulthood. A key role in the mechanisms of formation of memory and attention is played by the glutamatergic system. However, there has been virtually no systematic study on the effect of early postnatal stress on the expression of glutamatergic system genes in various regions of the brain in mice. In this study, we used two types of early postnatal stress: prolonged separation of pups from mothers (for 3 hours per day) and shortterm separation (15 minutes per day) during the first 2 weeks of life. We used an object recognition test to evaluate attention and memory to assess cognitive abilities in adults. We found that prolonged maternal separation reduced the ability to recognize a novel object and also disrupted motor and exploratory activities in adult animals, while short-term separation did not affect the studied parameters. We assessed the expression of the major genes of the glutamatergic system (AMPA receptor subunits Gria1, Gria2; NMDA subunits Grin1, Grin2a, and Grin2b; metabotropic receptor subunits Grm1, Grm2, and Grm3; glutamate transporters Vglut2, Eaat2, and Rab4a) in the frontal cortex, hippocampus, and hypothalamus. In the group with prolonged maternal separation, we found a decrease in the expression of Grin2b in the hypothalamus in comparison with the control, which led to a decrease in the mRNA ratio of this subunit to Grin2a mRNA, and possibly to a change in the ratio of these subunits in the NMDA receptor. In spite of the revealed cognitive impairments, we did not find significant changes in the expression of genes in the frontal cortex and hippocampus. Shortterm daily separation from mothers did not lead to changes in cognitive abilities and expression of genes of the glutamatergic system in mice. Thus, our results show that prolonged maternal separation may lead to a redistribution of the receptor subunits in the hypothalamus, which can modify the activity of the HPAA and determine the response to stress in these mice.     

Two major distinct subpopulations of neurokinin-3 receptor-expressing neurons in the superficial dorsal horn of the rat spinal cord

This study was designed to provide evidence for elucidating the mechanisms of neurokinin-3 receptor (NK3) in spinal pain modulation. First, colocalization of NK3 with the micro -opioid receptor (MOR1) was studied in the spinal dorsal horn of the rat. Confocal microscopy showed that about 44% of NK3-expressing neurons in laminae I and II were immunoreactive for MOR1, which corresponded to about 93% of the total population of MOR1-containing neurons in these laminae. Second, the relationship between NK3/MOR1-coexpressing neurons and those that express nitric oxide synthase (NOS) was examined by using a triple immunofluorescent staining method. About 37% of NK3-immunoreactive neurons were also NOS-immunoreactive, which constituted about 82% of NOS-immunoreacitve neurons in the superficial laminae. However, no triple-labelled neurons were detected. The present results indicate that there are two major distinct subpopulations of NK3-expressing neurons in the superficial dorsal horn, which suggests that the involvement of NK3 receptor in spinal nociception could be mediated by two distinct mechanisms, i.e. opioid and nitric oxide.     

Glutamate and metabotropic glutamate receptors associated with innervation of the uterine cervix during pregnancy: receptor antagonism inhibits c-Fos expression in rat lumbosacral spinal cord at parturition

Dorsal root ganglia (DRG) neurons connect the spinal cord and uterine cervix, and are activated at parturition with subsequent stimulation of secondary neurons in the spinal dorsal horn and autonomic areas. Neuropeptide neurotransmitters and receptors have been studied in these areas, but amino acid transmitters, e.g., glutamate, an excitatory neurotransmitter involved in sensory and nociceptive processing, have not been characterized. To determine if glutamate is involved in innervation of the cervix, rats were examined for markers of glutamatergic neurons in the L6-S1 spinal cord, DRG and cervix. Metabotropic glutamate receptors mGluR5 in the spinal dorsal horn and their expression over pregnancy were examined in pregnant rats and pregnant rats treated continuously with an antagonist of mGluR5, 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rats were allowed to deliver pups to determine if the antagonist altered the expression of an early response gene protein, Fos, in the L6-S1 cord. Immunohistochemistry showed glutamate- and vesicular glutamate transporter1 (VGluT1)-positive fibers in the cervix, glutamate- and VGluT1-expressing neurons in the DRG, some of which also exhibited retrograde tracer from cervical injections, and VGluT1 and mGluR5 immunoreactivities in the L6-S1 spinal dorsal horns. Expression of mGluR5 receptors increased over pregnancy. Fos-positive neurons were present among mGluR5-immunoreactivity in the spinal dorsal horn. Parturition-induced Fos-positive neurons in the spinal cords were abundant in control rats, but were reduced by 70% in MPEP-treated animals. These results suggest that glutamate is likely involved in the transmission of sensory signals, possibly pain, from the cervix to the spinal cord at parturition.     

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.     

Neuronal and glial localization of the cannabinoid-1 receptor in the superficial spinal dorsal horn of the rodent spinal cord

A long line of experimental evidence indicates that endogenous cannabinoid mechanisms play important roles in nociceptive information processing in various areas of the nervous system including the spinal cord. Although it is extensively documented that the cannabinoid‐1 receptor (CB₁‐R) is strongly expressed in the superficial spinal dorsal horn, its cellular distribution is poorly defined, hampering our interpretation of the effect of cannabinoids on pain processing spinal neural circuits. Thus, we investigated the cellular distribution of CB₁‐Rs in laminae I and II of the rodent spinal dorsal horn with immunocytochemical methods. Axonal varicosities revealed a strong immunoreactivity for CB₁‐R, but no CB₁‐R expression was observed on dendrites and perikarya of neurons. Investigating the co‐localization of CB₁‐R with markers of peptidergic and non‐peptidergic primary afferents, and axon terminals of putative glutamatergic and GABAergic spinal neurons we found that nearly half of the peptidergic (immunoreactive for calcitonin gene‐related peptide) and more than 20% of the non‐peptidergic (binding isolectin B4) nociceptive primary afferents, more than one‐third and approximately 20% of the axon terminals of putative glutamatergic (immunoreactive for vesicular glutamate transporter 2) and GABAergic (immunoreactive for glutamic acid decarboxylase; GAD65 and/or GAD67) spinal interneurons, respectively, were positively stained for CB₁‐R. In addition to axon terminals, almost half of the astrocytic (immunoreactive for glial fibrillary acidic protein) and nearly 80% of microglial (immunoreactive for CD11b) profiles were also immunolabeled for CB₁‐R. The findings suggest that the activity‐dependent release of endogenous cannabinoids activates a complex signaling mechanism in pain processing spinal neural circuits into which both neurons and glial cells may contribute.     

Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat

The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52 degrees C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Adelta and Abeta fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Abeta fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Abeta afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents-in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing.     

Postnatal phenotype and localization of spinal cord V1 derived interneurons

Developmental studies identified four classes (V0, V1, V2, V3) of embryonic interneurons in the ventral spinal cord. Very little is known, however, about their adult phenotypes. Therefore, we characterized the location, neurotransmitter phenotype, calcium-buffering protein expression, and axon distributions of V1-derived neurons in the adult mouse spinal cord. In the mature (P20 and older) spinal cord, most V1-derived neurons are located in lateral LVII and in LIX, few in medial LVII, and none in LVIII. Approximately 40% express calbindin and/or parvalbumin, while few express calretinin. Of seven groups of ventral interneurons identified according to calcium-buffering protein expression, two groups (1 and 4) correspond with V1-derived neurons. Group 1 are Renshaw cells and intensely express calbindin and coexpress parvalbumin and calretinin. They represent 9% of the V1 population. Group 4 express only parvalbumin and represent 27% of V1-derived neurons. V1-derived Group 4 neurons receive contacts from primary sensory afferents and are therefore proprioceptive interneurons. The most ventral neurons in this group receive convergent calbindin-IR Renshaw cell inputs. This subgroup resembles Ia inhibitory interneurons (IaINs) and represents 13% of V1-derived neurons. Adult V1-interneuron axons target LIX and LVII and some enter the deep dorsal horn. V1 axons do not cross the midline. V1-derived axonal varicosities were mostly (>80%) glycinergic and a third were GABAergic. None were glutamatergic or cholinergic. In summary, V1 interneurons develop into ipsilaterally projecting, inhibitory interneurons that include Renshaw cells, Ia inhibitory interneurons, and other unidentified proprioceptive interneurons.     

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.     

Subpopulations of cholinergic, GABAergic and glutamatergic neurons in the pedunculopontine nucleus contain calcium-binding proteins and are heterogeneously distributed

Neurons in the pedunculopontine nucleus (PPN) are highly heterogeneous in their discharge properties, their neurochemical markers, their pattern of connectivity and the behavioural processes in which they participate. Three main transmitter phenotypes have been described, cholinergic, GABAergic and glutamatergic, and yet electrophysiological evidence suggests heterogeneity within these subtypes. To gain further insight into the molecular composition of these three populations in the rat, we investigated the pattern of expression of calcium binding proteins (CBPs) across distinct regions of the PPN and in relation to the presence of other neurochemical markers. Calbindin- and calretinin-positive neurons are as abundant as cholinergic neurons, and their expression follows a rostro-caudal gradient, whereas parvalbumin is expressed by a low number of neurons. We observed a high degree of expression of CBPs by GABAergic and glutamatergic neurons, with a large majority of calbindin- and calretinin-positive neurons expressing GAD or VGluT2 mRNA. Notably, CBP-positive neurons expressing GAD mRNA were more concentrated in the rostral PPN, whereas the caudal PPN was characterized by a higher density of CBP-positive neurons expressing VGluT2 mRNA. In contrast to these two large populations, in cholinergic neurons expression of calretinin is observed only in low numbers and expression of calbindin is virtually non-existent. These findings thus identify novel subtypes of cholinergic, GABAergic and glutamatergic neurons based on their expression of CBPs, and further contribute to the notion of the PPN as a highly heterogeneous structure, an attribute that is likely to underlie its functional complexity.     

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions

Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron-to-glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up-regulated in a regionally specific manner in response to neuropathy. CX3CR1-expressing cells were identified as microglia by their cellular morphology and positive OX-42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.     

Morphology and electrophysiological properties of hamster spinal dorsal horn neurons that express VGLUT2 and enkephalin

The excitatory amino acid glutamate mediates transmission at spinal synapses, including those formed by sensory afferent fibers and by intrinsic interneurons. The identity and physiological properties of glutamatergic dorsal horn neurons are poorly characterized despite their importance in spinal sensory circuits. Moreover, many intrinsic spinal glutamatergic synapses colocalize the opioid peptide enkephalin (ENK), but the neurons to which they belong are yet to be identified. Therefore, we used immunohistochemistry and confocal microscopy to investigate expression of the VGLUT2 vesicular glutamate transporter, an isoform reported in nonprimary afferent spinal synapses, and ENK in electrophysiologically identified neurons of hamster spinal dorsal horn. VGLUT2 immunoreactivity was localized in restricted fashion to axon varicosities of neurons recorded from laminae II-V, although the occurrence of immunolabeling in individual varicosities varied widely between cells (39 +/- 36%, n = 31 neurons). ENK colocalized with VGLUT2 in up to 77% of varicosities (17 +/- 21%, n = 21 neurons). The majority of neurons expressing VGLUT2 and/or ENK had axons with dense local terminations or projections consistent with propriospinal functions. VGLUT2 and ENK labeling were not correlated with cellular morphology, intrinsic membrane properties, firing patterns, or synaptic responses to sensory afferent stimulation. However, VGLUT2 expression was significantly higher in neurons with depolarized resting membrane potential. The results are new evidence for a population of dual-function dorsal horn interneurons that might provide another mechanism for limiting excitation within dorsal horn circuits during periods of strong sensory activation.     

Anatomical characterization of the cannabinoid CB1 receptor in cell‐type–specific mutant mouse rescue models

Type 1 cannabinoid (CB₁) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB₁ receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell‐type–specific rescue of CB₁ receptors have emerged as excellent tools for investigating CB₁ receptors’ cell‐type–specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB₁ receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB₁ receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu‐CB₁‐RS) or GABAergic neurons (GABA‐CB₁‐RS) was studied by immunoelectron microscopy. Results were compared with conditional CB₁ receptor knockout lines. As expected, CB₁ immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB₁ receptor‐immunopositive excitatory and inhibitory synapses were Glu‐CB₁‐RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA‐CB₁‐RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB₁ receptor‐immunopositive excitatory and inhibitory synapses in the inner one‐third of the dentate molecular layer was Glu‐CB₁‐RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA‐CB₁‐RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu‐CB₁‐RS and GABA‐CB₁‐RS mice show the usual CB₁ receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in‐depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302–318, 2017. © 2016 Wiley Periodicals, Inc.