VGLUT-VGAT expression delineates functionally specialised populations of vasopressin-containing neurones including a glutamatergic perforant path-projecting cell group to the hippocampus in rat and mouse brain

Abstract

The origin and functional significance of vasopressin (AVP)-containing fibres in limbic regions has been an ongoing subject of investigation for several years. We have previously identified AVP-magnocellular neurones of rat hypothalamus that provide glutamatergic projections to the hippocampus, amygdala, lateral habenula and locus coeruleus. However, we also reported AVP-immunopositive fibres in those regions that are thin and make Gray type II synapses, which are unlikely to be of magnocellular origin. Therefore, in the present study, we characterised AVP mRNA co-expression with expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) neuronal traits in rat and mouse brain, using high-resolution in situ hybridisation methods, including a radio-ribonucleotide and RNAscope 2.5 HD duplex assay, with Slc17a7, Slc17a6, Slc32a1 and Avp probes corresponding to mRNAs of VGLUT1, VGLUT2, VGAT and AVP, respectively. We located 18 cell groups expressing Avp and identified their molecular signatures for VGLUT and VGAT mRNA expression. Avp cell groups of hypothalamus and midbrain are mainly VGLUT mRNA-expressing, whereas those in regions derived from cerebral nuclei are mainly VGAT mRNA-expressing, suggesting a functional segregation of glutamate/GABA co-transmission with AVP. A newly identified Slc17a7 and Slc17a6 (but not Slc32a1) expressing vasopressinergic cell group was found in layer II-III neurones of the central entorhinal cortex, which projects to the hippocampus. These data support the notion of a complex role for AVP with respect to modulating multiple central circuits controlling behaviour in specific ways depending on co-transmission with glutamate or GABA, potentially giving rise to a functional classification of AVPergic neurones in the central nervous system.

1 |. INTRODUCTION

With the advent of intersectional genetics for manipulating the function of specific neuronal populations, as well as optogenetics for exploring their roles within defined functional circuits, neuroscience has entered a new phase in the exploration of central nervous system (CNS) neurotransmission. Neuropeptides have become important markers, and their promoters important tools, in this enterprise,^1–3 helping to define circuits and subcircuits for the specific control of behaviours as complex as nocifense, parenting and fear responding. An underexplored facet of neurotransmission exposed by such studies is the extent to which neuropeptides are important in transmission, including as regulators of the expression of other transmitters, and also as co-transmitters conferring unique post-synaptic properties to GABAergic and glutamatergic neurones. A first step towards addressing these important issues is to clearly identify subpopulations of peptidergic neurones in circuits for which the physiological and electrophysiological properties of peptide-receptor signalling are well known, as well as determine how they differ in their co-expression of glutamate and GABA. This information is critical for parsing the post-synaptic actions of peptides on this specific neurochemical basis.⁴

Vasopressin neurones of the mammalian brain are an obvious target for further classification by a microneurochemical approach. It is well known that parts of the vasopressinergic magnocellular system express genes and proteins necessary for both glutamate and vasopressin neurotransmission.^5,6 Release at the nerve terminals of the posterior pituitary is functionally critical for the hormonal actions of vasopressin itself at peripheral receptors; however, no clear role for glutamate secretion at this site.⁷ This raises the question of whether glutamate co-transmission is of actual physiological importance in these neurones, either at their CNS or their pituitary projections, or whether the expression of glutamatergic traits by these neurones serves some intracellular or autocrine rather than neurotransmitter function.

The origin of vasopressin (AVP)-containing afferents to given limbic regions, including the hippocampus, has been the subject of detailed recent investigations. AVP-magnocellular neurones of the hypothalamus provide glutamatergic projections to the hippocampus,^8,10 amygdala,¹¹ lateral habenula^12,13 and locus coeruleus.¹⁴ However, we have also reported AVP-immunopositive fibres in those regions, which are much thinner and make Gray type II synapses. The origins of such fibres and their role in circuit interaction are unclear. In the present study, we first provide additional evidence of the existence of mixed AVP-containing fibres in the rat hippocampus at the electron microscopic level, augmenting our previous study.⁸ We then characterise AVP co-expression with glutamatergic and GABAergic markers at the mRNA level in rat and mouse brain, using high-resolution in situ hybridisation methods, including a radio-ribonucleotide and RNAscope^® 2.5 HD duplex assay, with vesicular glutamate transporter VGLUT1 (Slc17a7), VGLUT2 (Slc17a6), vesicular GABA transporter (VGAT) (Slc32a1) and AVP probes. We found 17 cell groups expressing AVP that possess unique molecular signatures with respect to VGLUTs and/or VGAT expression. From an ontological perspective, the AVP cell groups derived from the hypothalamus, cerebral cortex and midbrain are mainly VGLUT- mRNA expressing, whereas the structures derived from cerebral nuclei (striatum and pallidum¹⁵) are mainly VGAT- mRNA expressing, suggesting a functional segregation of this co-transmission phenomenon within the AVP brain signalling network. Finally, the identification of a previously unidentified AVP projection from entorhinal cortex, including its potential functional role(s), is described.

2 |. MATERIALS AND METHODS

2.1 |. Animals

Eight male Wistar rats and eight male C57Bl6N mice, all young adults (around 2 months old) from a local animal breeding facility were used in the present study. All procedures were approved by the Research and Ethics Committees of the Faculty of Medicine, Universidad Nacional Autónoma de México (CIEFM-062–2016) and Animal Care and Use Committee, NIMH-IRP, in accordance with the principles stated in the Handbook for the Use of Animals in Neuroscience Research (Society for Neuroscience. Washington, DC 1991 and as updated periodically).

2.2 |. Immunohistochemistry for transmission electron microscopy

Immunoelectron microscopy procedures were performed as reported previously.⁸ Briefly, rats were deeply anaesthetised with sodium pentobarbital (63 mg kg⁻¹, Sedalpharma) and then perfused first with 0.9% saline, followed by a fixative containing 4% paraformaldehyde, 15% v/v saturated picric acid and 0.05% glutaraldehyde in 0.1 mol L⁻¹ sodium phosphate buffer (PB, pH 7.4) for 15 minutes. Coronal sections (70 μm) containing hippocampus were selected. Non-specific antibody binding was blocked with 20% normal swine serum (NSS) in Tris-buffered saline (TBS) + Triton-X-100 0.025% for 1 hour. Sections were then incubated with a cocktail of two rabbit anti-arginine vasopressin antibodies (dilution 1:2000, gift from Professor R. M. Buijs, UNAM, Mexico;⁹ dilution 1:5000, T4563, Peninsula Laboratories Inc., Belmont, CA, USA) in TBS plus 1% NSS for 48 hour at 4°C with gentle shaking, and rinsed and incubated with secondary antibody (swine anti-rabbit IgG conjugated with horseradish peroxidase; P021702, dilution 1:100, Dako, Glostrup, Denmark) in TBS containing 1% NSS, overnight at 4°C. Peroxidase enzyme reaction was carried out using the chromogen 3,3′-diaminobenzidine (0.05%; Electron Microscopy Sciences, Hatfield, PA, USA) and hydrogen peroxide (H₂O₂, 0.01%) as substrates. Sections were post-fixed with 1% osmium tetroxide in 0.1 mol L⁻¹ PB for 1 hour and dehy-drated through a series of graded alcohols (including 45 minutes of incubation in 1% uranyl acetate in 70% ethanol), then transferred to propylene oxide, followed by Durcupan ACM epoxy resin (Electron Microscopy Sciences). Sections were flat-embedded on glass microscope slides, and the resin was polymerised at 60°C for 2 days. Areas containing AVP-immunolabelled axons in the medial part of the lateral habenula were re-embedded in capsules with Durcupan resin. Ultrathin sections (approximately 70 nm) were cut with an ultramicrotome using a diamond knife. Sections were collected onto pioloform-coated single slot grids and examined with a CM100 transmission electron microscope (Philips, Eindhoven, The Netherlands). Digital electron micrographs were obtained with a digital micrograph 3.4 camera (Gatan, Inc., Pleasanton, CA, USA) and scaled with imagej (NIH, Bethesda, MD, USA) and photoshop (Adobe Systems Inc. San Jose, CA, USA).

2.3 |. Riboprobe in situ hybridisation procedure

Detailed methods are described elsewhere.¹⁶ Briefly, Wistar rats were deeply anaesthetised with sodium pentobarbital (Sedalpharma, 63 mg kg⁻¹ body weight, i.p.) and perfused via ascending aorta with 0.9% saline followed by cold fixative containing 4% of paraformaldehyde in 0.1 mol L⁻¹ PB, pH 7.4. Brains were post-fixed with 1% paraformaldehyde in PB and kept at 4°C until use. Serial sagittal sections were obtained by cryosectioning (12 μm) of whole rat brain using a Leica CM1950 cryostat (model 35578; Leica Microsystems, Wetzlar, Germany). In situ hybridisation (ISH) was performed in one in six sections as described previously¹⁷ using ³⁵S- and ³³P-UTP labelled ribonucleotide probes. The pT7T3D-PacI plasmid (accession number: AI072073, clone ID: 1786383; Thermo Scientific, Waltham, MA, USA) containing rat arginine vasopressin cDNA (602 bp, accession number: NM_016992) was linearised with EcoRI and then transcribed in vitro with T3 RNA polymerase to yield antisense complementary RNA probe. The construct was verified by sequencing. The radioactivity was adjusted to 10⁷ cpm mL⁻¹ hybridisation buffer. Sections were mounted on coated slides, air-dried. Slides were first exposed to autoradiography film and analysed on a phosphorimager (BAS5000; Fuji, Tokyo, Japan) and then dipped in nuclear track emulsion (Eastman Kodak, Rochester, NY, USA), and exposed for 4 weeks prior to development. Slides were counterstained with methylene blue for histological examination.

2.4 |. RNAscope duplex in situ hybridisation procedure

Rats (male, Wistar, n = 4) and mice (male, C57Bl6, n = 8) were used for the present study. To confirm the observations obtained from radio-riboprobe method described above, and to identify the glutamatergic vs GABAerigc nature of the cell groups, we performed a RNAscope duplex ISH procedure with two different protocols: (i) in accordance with the manufacturer’s instructions for fresh-frozen rodent brain tissue for the manual chromogenic assay (RNAscope 2.5 HD duplex reagent kit) (https://acdbio.com/technical-support/user-manuals), for n = 2 and n = 4, rats and mice respectively, and (ii) modifying the fixed-frozen protocol outlined by the manufacturer according to Mongia et al¹⁸ to reduce possible nucleic acid degradation during the preparation. Both methods yielded qualitatively similar results.

For the first procedure, two rats and four mice were deeply anaesthetised and decapitated using a small animal guillotine (Kent Scientific Corp., Torrington, CT, USA). Brains were removed and rapidly frozen in dry ice powder. The fresh-frozen tissue sections (12 μm) were obtained using a Leica CM1520 cryostat and mounted on positively-charged glass slides (Thermo Scientific). The probes for in situ hybridisation used to identify the transcripts for Slc32a1, Slc17a6, Slc17a7, Slc17a8 and AVP were designed and provided by Advanced Cell Diagnostics (Newark, CA, USA). All experimental steps were performed in accordance with the manufacturer’s instructions for fresh-frozen sections.

For the second procedure, the animals were transcardially perfused with 4% paraformaldehyde in DEPC treated 0.1 mol L⁻¹ phosphate-buffered saline (PBS) (PBS tablets, P-4417; Sigma, St Louis, MO, USA). Brains were post-fixed in paraformaldehyde for 2 hours at 4°C, rinsed with PBS and transferred to solutions of 12%, 14% 16% and 18% sucrose in PBS (0.1 mol L⁻¹). The solutions were changed when the brains sank to the bottom of the solution vial. After cryoprotection, brains were embedded in OCT and frozen in powdered dry ice. Sagittal sections (12 μm) were mounted onto SuperFrost slides (Thermo Scientific) and dried for 1 hour at 60°C. Slides were then treated with target retrieval reagents at 100°C for 5 minutes and protease-plus digestion for 15 minutes, followed by hybridisation with duplex probes and amplification steps.

3 |. RESULTS

3.1 |. The hippocampus, a limbic region, has three types of AVP-immunopositive afferents of apparently extra-hippocampal origin

We have previously reported that, in the hippocampus⁸ and amygdala¹¹, there are two types of AVP-immunopositive fibres: thick ones (with an average thickness of 700–1000 nm) and thin ones (with an average thickness of 200–500 nm), distributed and intermingled in selective subfields, with both selectively establishing Gray I and Gray type II synapses preferentially at pyramidal neurones and interneurones, respectively.⁸ In the present study, we report an additional observation of a third type that made excitatory synapses onto unidentified hippocampal cellular segments. Figure 1A provides an example of two previously reported types of AVP+ fibres in the striatum radiatum of ventral hippocampus CA2. The schematic drawing (Figure 1B) symbolises the two classical types of synapses: the Gray type I, which is generally associated with glutamatergic synapses, and the Gray type II, which is generally associated with GABAergic synapses. Here, we report three types of axons at the electron microscopy level. The first type, which will be referred to as type A, (Figure 1C) has a thick axon (diameter of approximately 700 nm), full of AVP-immunopositive (AVP+) dense core vesicles (dcv). This type of fibre establishes Gray type I synapses (Figure 1C, inset, with post-synaptic density indicated by a green arrowhead). Examples of type A axons, and some of the post-synaptic components identified, are provided found in the previous study.⁸ The second type, which had not been reported previously, and is referred to here as type B, has an intermediate axon diameter and many small clear synaptic vesicles intermingled with few large AVP+ dcv at its axon terminals (Figure 1D). Type B fibres also establish Gray type I synapses (Figure 1D, inset, taken from the ventral hippocampal CA1, stratum radiatum and the green profile in Figure G). A third type, type C, features AVP+ thin fibres,⁸ which establish Gray type II synapses onto hippocampal neurones (Figure 1E and Figure 1E, inset; see also the pink profiles of Figure F and G, which are the same profiles in two neighbouring sections displaying both the immunoreactivity to AVP (AVP+) (Figure F) and the synapse established by the AVP+ axons; see also the previous study⁸).

FIGURE 1.

Three types of vasopressin-immunopositive (AVP+) fibres are present in the ventral hippocampus of the rat, suggesting three different functionally specialised afferent regions for its modulation. A, An example of the thick and thin AVP+ fibres in the stratum radiatum (str. rad.) of ventral hippocampus (VHi) CA2 region. B, Schematic drawing to symbolise the two classical types of synapses, Gray type I, with presence of post-synaptic density (PSD), indicated with a single green arrowhead, and Gray type II, in the absence of PSD and indicated with two red arrowheads). C, D, E, The three AVP+ fibre-types reported in the Results, (A), (B), (C), and examples of their corresponding synapses. F, G, Adjacent sections of the same profile illustrate the Gray type I and Gray type II synapses established by AVP+ axons onto a dendrite. F, The AVP+ dense core vesicles (dcv) can be seen in the pink profile and in (G) both the pink and green profiles (also AVP+) established Gray type I and Gray type II synapses onto the same unidentified dendrite. Scale bars = 500 nm unless specified otherwise. The photograph in (A) and the the inset in (C) are modified with permission.⁸ Scale bars = 500 nm, except in (A)

3.2 |. Eighteen arginine vasopressin-expressing cell regions identified in rodent brain

With the two high-resolution ISH methods, we identified a total of 18 regions that expressed AVP mRNA and could then be further examined for excitatory or inhibitory neurotransmitter expression traits. In all regions evaluated with the three methods used in the present study, we obtained similar results with respect to the distribution pattern of AVP and their molecular signatures regarding glutamatergic or GABAerigic cell types, although the modified double RNAscope protocol (vide supra) yieded better tissue preservation.

Figure 2 depicts the serial sagittal sections (representative) of young adult rat brain (n = 4) that were hybridised with antisense radioactive AVP riboprobe. Autoradiographs with overnight exposure were read by a phosphorimager. Seventeen regions, ordered numerically, from medial to lateral, are shown in Figure 2 with schematic drawings illustrating the cell groups expressing AVP. Previously reported cell groups were confirmed,^19,20 in addition to three regions, zona incerta (ZI) of hypothalamus, ventral tegmental area (VTA) of midbrain and entorhinal cortex layer II, which, to the best of our knowledge, have not been reported (Table 1).

FIGURE 2.

Distribution of vasopressin mRNA (AVP) expression in rat brain: 18 AVP-expressing regions of rat brain were revealed by antisense radioactive AVP riboprobe (autoradiographs with overnight exposure were read by phosphorimager). To facilitate recognition of these anatomical regions in autoradiographic sagittal plates (labelled from A to L in a septo-temporal order), schematic drawings of each of the plates are shown below based on anatomical observation with reference to the Rat Brain Atlas of Swanson,¹⁵ with the AVP-expressing regions numbered as they appear in a medial-lateral order. Note that in (I), (J), (K) and (L) layer II of entorhinal cortex, central and lateral subdivisions were all clearly positive to AVP mRNA. Abbreviations: BLA: basolateral amygdala; inf stem : infundibular stalk; see Table 1 for the abbreviations of the numbered regions.

TABLE 1.

Co-localisation of vasopressin (Avp)-expressing nuclei with the mRNA for glutamatergic and GABAergic transporters^b

Brain regiona	Abbreviation	Avp (AVP)d	Slc17a7 (VGLUT1)e	Slc17a6 (VGLUT2)e	Slc32a1 (VGAT)e	n.c.l.e,f
Hypothalamic region
Paraventricular nucleus1
Medial magnocellular	PVNMM	++++	n.o.c	++	n.o	++
Medial parvicellular dorsal	PVNMPD	+++	n.o.	+	n.o	++
Lateral magnocellular dorsal	PVNLMD	+++++	n.o.	++++	n.o	+
Accessory nuclei4	AN	++++	n.o.	+++	n.o.	+
Supraoptic nucleus3	SON	+++++	n.o	+++	+	+
Suprachiasmatic nucleus2	SCN	++++	n.o.	n.o	+++	++
Lateral hypothalamus8	LH	+	n.o.	+	n.o.	-
Zona incerta6	ZI	+	n.o.	+	n.o	-
Midbrain
Ventral tegmental area10	VTA	+	n.o.	+	+	-
Cortex
Entorhinal cortex (L-II, projection neurone)
Lateral15	LEnt	++	++	++	n.o.	-
Central16	CEnt	++	++	++	n.o.	-
Main olfactory bulb17	MOB	++	+	+	n.o.	-
Anterior olfactory nucleus18	AON	+	+	+	+	-
Piriform cortex19	PC	++	+	+	n.o.	-
Cerebral nuclei
Bed nucleus of stria terminalis
Postero-lateral7	BNSTpl	++	n.o.	n.o.	+	+
Antero-medial5	BNSTam	+	n.o.	n.o.	+	+
Amygdaloid complex
Intra-amygdaloid division of the BNST13	STIA	++	n.o.	n.o.	++	-
Medial amygdala, antero-ventral11	MeAAV	++	n.o.	n.o.	++	-
Medial amygdala, postero-dorsal12	MeAPD	++	n.o.	n.o.	++	-
Central amygdala14	CeA	+	n.o.	n.o.	+	-

^aOrganisation of brain regions based on the Swanson Atlas of the Rat Brain.¹⁵

^bSemiquantitative description of Avp-expressing nuclei that co-express the mRNA coding for the vesicular glutamate transporters (Slc17a7 for VGLUT1 and Slc17a6 for VGLUT2) or GABA transporter (slc32a1).

^cn.o.: not observed.

^dThe number of Avp-expressing nuclei over the total number of Nissl stained nuclei was quantified. 81%−100%, +++++; 61%−80%, ++++; 41%−60%, +++, 21%−40%; ++, 1%−20%, +.

^eThe number of + indicates the proportion of nuclei positive for VLGUT1, VGLUT2 and VGAT mRNA with respect to the total number of Avp-expressing nuclei.

^fn.c.l. Not co-localised with any of the three vesicular transporters RNAs.

Bold letters indicate regions where we found Avp expression.

Specifically, a newly observed AVP mRNA -expressing cortical region, layer II of the entorhinal cortex in its central to lateral extension, was revealed to express AVP with the radio-riboprobe experiment and confirmed with the two RNAscope methods decribed above (Figure 3). These AVP mRNA -expressing neurones co-expressed Slc17a6 and Slc17a7, mRNAs that encode the vesicular glutamate transporters II and I, respectively (Figure 3E, F), although they did not overlap with Slc32a1, mRNA that encodes the vesicular GABA transporter (Figure 3G). The expression of Avp in this region is apparently higher in rat compared to mice from the samples we examined (data not shown), although we refrain from providing a quantitative affirmation as a result of the crucial influence of individual experimental procedures using this manual chromogenic assay. In other words, each experimental procedure for each subject can produce variable observations not necessarily in proportion to the animals’ intrinsic properties but, instead, influenced by given experimental processing. However, we found most Avp expressing neurons did not express Slc32a1 (mRNA for VGAT)

FIGURE 3.

The newly documented vasopressin (AVP)-expressing neurones in pyramidal layer II of entorhinal cortex, in both rat and mouse, co-expressed slc17a6 and Slc17a mRNAs encoding the vesicular glutamate transporters II and I respectively. A, Low magnification bright-field photomicrographs of one representative sagittal section (rat) from Ilford K.5 nuclear tract emulsion dipped slides exposed in the dark at 4°C for 4 weeks. The layer II pyramidal neurones expressing AVP mRNA can be clearly observed. Sections were counterstained with methylene blue. B, Schematic drawing based on the microscopic observation with reference to the Rat Brain Atlas of Paxinos and Watson (³²)). C, D, Photomicrographs of higher magnifications of layer II of entorhinal cortex of the rat. E, F, RNAscope duplex labelling of mouse brain showing the co-expression of AVP with Slc17a7 and Slc17a6, respectively. G, No co-expression of AVP and Slc32a1 (a mouse brain is shown). Scale bars: (A) 400 μm; (C) 50 μm; (D) 10 μm; (E, F, G): 20 μm. Abbreviations: CA1: Cornu Ammonis field 1, Cereb: cerebellum, DG: dentate gyrus, EC: enthorinal cortex, MG: medial geniculate nucleus, Sub: subiculum, VC: visual cortex

A third type of vasopressin-containing fibre (type B), containing mainly small clear synaptic vesicles and few large AVP immunopositive dcv, was observed making Gray type I synapses in the ventral CA1, stratum radiatum. This finding fits well with the observation that layer II pyramidal neurones of the entorhinal cortex, representing the origin of the perforant path, express Avp in the rat.

To identify the molecular (glutamatergic or GABAergic) signature of each Avp-expressing cell group, we performed a RNAscope 2.5 HD duplex assay, with VGLUT1, VGLUT2 and VGAT, together with AVP probes in mouse and rat brain. No significant differences were found between these two rodent species, with the exception of the possible quantitative differences in entorhinal cortex, as discussed above.

Table 1 shows the VGLUT and VGAT co-expression analysis of these 18 regions. Generally speaking, the Avp-expressing structures belonging to the hypothalamus, midbrain and cortex were predominantly glutamatergic, whereas the Avp-expressing structures derived from cerebral nuclei (striatum and pallidum) were predominantly GABAergic.

Figure 4 provides particularly noteworthy examples of Avp co-expression with Slc17a7, Slc17a6 and Slc32a1, mRNAs for VGLUT1, VGLUT2 and VGAT respectively. Figure 4A and 4B depicts the coronal plate of hypothalamus where the three main Avp-expressing nuclei were observed with Avp/Slc17a6 (VGLUT2, 4A) and Avp/Slc32a1 (VGAT, 4B) probes. In the lateral subdivision of the paraventricular nucleus, around four-fifths of the AVP mRNA+ nuclei strongly co-expressed Slc17a6 mRNA (Figure 4A and 4C, paraventricular nucleus [PVN], double arrowheads), although the Avp-expressing nuclei toward the ventricle lacked co-expression of Slc17a6 (indicated by single green arrows). Both medial and lateral subdivisions of PVN lacked Slc32a1 expression (Figure 4B, green arrows and Figure D). In the supraoptic nucleus, about three-fifths of the Avp-expressing nuclei co-expressed Slc17a6 (VGLUT2) (Figure 4E), one-fifth without co-localisation. Surprisingly, some of the Avp-expressing cells in the rostral region of the supraoptic nucleus (SON) co-expressed Slc32a1 (VGAT) (Figure 4F), although note that these are untested for VGLUT2 expression as a result of limitations in co-labelling with the ISH technique employed.

FIGURE 4.

Examples showing vasopressin (AVP) co-expression with glutamate or GABA markers in some brain regions (samples taken from mouse brain). In situ hybridisation using RNAscope 2.5 HD duplex assay, with Slc17a7 (vesicular glutamate transporter, VGLUT1), Slc17a6 (VGLUT2) and Slc32a1 (vesicular GABA transporter, VGAT) probes in combination with AVP probe. A, B, Coronal plane of hypothalamus where the three main Avp-expressing nuclei were observed with Avp/Slc17a6 (VGLUT2, A) and Avp/Slc32a1 (VGAT, B). A particular feature detected with the RNAscope technique is that some of the hypothalamic AVP cell main axons were labelled by the AVP mRNA probe (A’). In the paraventricular nucleus, the lateral subdivision strongly co-expressed Avp+/Slc17a6+, as indicated by double red/green arrowheads; see also (C), although the Avp-expressing cells toward the ventricle lacked co-expression of Slc17a6 (green arrows). Both medial and lateral subdivisions of PVN lacked expression of Slc32a1 (green arrows, D). In the supraoptic nucleus Avp-expressing nuclei co-expressed Slc17a6 (VGLUT2; A, E). The Avp cells in the suprachiasmatic nucleus (SCN) lacked mRNA for the glutamate transported and expressed the mRNA for VGAT (Slc32A1, B and B’). Some Avp-expressing cells in the rostral region of SON expressed Slc32a1 (VGAT, F). The main olfactory bulb (MOB) hosts a large population of Avp/Slc17a7 expressing cells in the periglomerular layer (G, double green/red arrowhead). The inset shows a high magnification photomicrograph where the double expression nature can be clearly distinguished in a low Avp expressed (green dots) Slc17a7 expressing cell. VGAT expression AVP+ cells in the bed nucleus of stria terminalis, postero-lateral division (BNSTpl) and central amygdala (CeA) is shown in (H) and (I), respectively. Abbreviations: BNSTpl: posterolateral div. of the bed nucleus of stria terminalis, CeA: central amygdala, MOB: medial olfactory bulb, PVN: paraventricular nucleus, opt: optical tract, SCN: suprachiasmatic nucleus, SON: supraoptic nucleus

Another particular feature detected using the RNAscope technique is that some of the hypothalamic AVP-expressing cell’s main axons were labelled with the AVP probe (Figure 4A, inset). The radio-riboprobe method confirms that AVP is strongly present in the medial eminence (ME) and the infundibular stalk (Figure 2A).

Figure 4G illustrates the glutamatergic nature of the Avp-expressing periglomerular cells in the main olfactory bulb (MOB). VGAT expression in Avp+ cells in the bed nucleus of stria terminalis, postero-lateral division and central amygdala is shown in Figure 4H and I.

In summary, 18 groups of potential vasopressinergic neurones, widely distributed in the rodent CNS, can be distinguished on the basis of differential expression of markers for the glutamatergic versus GABAergic chemotype. Figure 5 comprises a schematic drawing summarising the findings of the present study.

FIGURE 5.

Schematic drawing summarising the features of the 18 vasopressin (AVP)-expressing cells common to both rats and mice, depicting their main molecular signature as glutamatergic (symbolised as green spheres) or GABAergic (symbolised as red spheres). See Table 1 for the abbreviations of the numbered regions. Pink shade region emphazises the newly discovered gluatamatergic cortical cell group projecting via perforant path to hippocampus in rodent brain

4 |. DISCUSSION

Investigation of the control and modulation of behaviour by central vasopressinergic neurones is an emerging area of neuroscience research in which anatomical, genetic, cellular plasticity and electrophysiological data are rapidly being acquired and integrated. Central vasopressin neurotransmission has been causally linked to the expression of anti-social behaviours such as aggression and social anxiety.²¹ Linkages between gonadal state and hydromineral status and these behaviours are being clarified as the full neurochemical potential of the ‘vasopressinergic’ neurone is becoming better characterised.^12,13,22 One way to gain further insight into how vasopressinergic neurones at various anatomical locations integrate physiological status with behaviour is to determine the actual distribution of vasopressin/excitatory and vasopressin/inhibitor neurones in magnocellular and parvocellular vasopressinergic neurones of the brain.

There have been several comprehensive chemoanatomical studies of central AVP neurone distribution.^20,23–26 However, the cell-type and brain circuit interaction of vasopressinergic neurones have not been comprehensively investigated at these various sites. Using high-resolution ISH methods, including a radio-ribonucleotide and RNAscope 2.5 HD duplex assay, with VGLUT1, VGLUT2, VGAT and AVP probes, we investigated the co-transmission signatures of vasopressinergic neurones throughout the CNS of the rat and mouse. We identified 18 AVP cell groups in both rat and mouse and we found a clear logic regarding the glutamatergic vs GABAergic molecular signatures that could be linked to their developmental origins and functions: the predominantly glutamatergic chemotype for olfaction (MOB, anterior olfactory nucleus, piriform cortex), learning and memory (entorhinal cortex to performant path), and homeostatic/allostatic regulation (PVN, SON), as well as the GABAergic chemotype for brain state (suprachiasmatic nucleus, SCN) and emotional modulation (antero-ventral medial amygdala, central amygdala, bed nucleus of stria terminalis).

The cytological classification of vasopressinergic neurones in the CNS in the last four decades has been established within three frames of reference. These are somatic size (magnocellular vs parvocellular); projection to the neural lobe of the pituitary gland or to the brain; and location of cell bodies either in the classical ‘hormonal’ nuclei of the hypothalamus, such as the PVN, SCN and SON, or within small cell groups at extrahypothalamic locations.^19,20,26,27 Recent findings revealed that the ascending projections of the magnocellular neurosecretory neurones may have profound pro-motivational and goal-directed effects projecting monosynaptically to the hippocampus, lateral habenula, amygdala and locus coeruleus in rats under conditions of physiological stress.^{11,14,28–30} Yet the role(s) of co-released classical transmitters in allowing or aiding these profound effects remain understudied, in part because a detailed census of the neurochemistry of these neurones has not been completed. Because the role of AVP in homeostatic vs allostatic regulation may depend on the location of the neurone, its integration in a given circuit(s) and the co-release of other transmitters, obtaining the molecular signatures these classical neurotransmission systems is therefore an important goal.

As summarised in Table 1, AVP neurones present a constellation of cell clusters, embedded in key anatomical circuits, with profound differences in the potential to affect these circuits based on co-transmission of GABA and glutamate. With this preliminary neurochemical atlas in hand, intersectional genetic approaches should yield a better understanding of the direct neurotransmitter and the classical transmitter post-synaptic modulatory roles of AVP in CNS.

In summary, the present study has addressed what constitutes a vasopressinergic neurone in terms of its essential classical co-transmitters. It is increasingly obvious that neuropeptides in general derive their special roles in behavioural adaptation via coordinated co-release with either an inhibitory or excitatory transmitter. Here, we have used ISH and a RNAscope procedure to catalogue the types of AVP neurones in the brain according to their co-transmitter and identify a basis for further functional sub-classification of the 18 AVP cell groups of the brain. In the process, we have discovered a new cell cluster in the entorhinal cortex present in rats and mice, although apparently it is more prominent in the former. The entorhinal cortex is the source of the perforant path that projects monosynaptically to the dentate gyrus, hippocampus proper and other cortical regions, such as the temporal cortex. An earlier study showed that the transection of the EC-temporal cortex pathway attenuated the vasopressin-induced improvement of memory in rats.³¹ To our knowledge, the present study is the first to report this new vasopressinergic/glutamatergic hub for memory in two phylogenetically related but different rodent species.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.